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Neuroscience Research 61 (2008) 249–256

Neuroprotective role of angiotensin II type 2 receptor after

transient focal ischemia in mice brain

Nobukazu Miyamoto, Ning Zhang, Ryota Tanaka, Meizi Liu,Nobutaka Hattori, Takao Urabe *

Department of Neurology, Juntendo University School of Medicine 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan

Received 25 December 2007; received in revised form 5 March 2008; accepted 17 March 2008

Available online 27 March 2008

Abstract

This study assessed the time course of angiotensin (Ang) II type 1 and type 2 receptor expression after 60 min of ischemia/reperfusion in mice

treated with a nonhypotensive dose of valsartan, an angiotensin II type 1 receptor antagonist. We also examined the potential neuroprotective

mechanisms mediated by angiotensin II type 2 receptor. Mice were divided into two groups (n = 64, each): valsartan-treated and control, vehicle

groups. Infarct volume and neurological deficit scores were evaluated at several time points after ischemia, while immunohistochemical analyses

were performed at serial time points after reperfusion. Valsartan significantly reduced the infarct volume and improved the neurological deficit

scores (P < 0.05). Both angiotensin II type 1 and type 2 receptors were upregulated at 24 h and peaked at 72 h with type I receptors dominating in

the ischemic penumbra of the vehicle group. Interestingly, angiotensin II type 2 receptor expression levels were significantly higher in the valsartan

group than vehicle controls (P < 0.001). Moreover, angiotensin II type 2 receptor upregulated phosphosignal transducer and activator of

transcription-3, and B-cell lymphoma protein-2 (P < 0.05). Our results indicated that angiotensin II type 2 receptor has antiapoptotic activity by

activating the B-cell lymphoma protein-2 via the janus kinase/signal transducer and activator of transcription signaling pathway.

# 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Keywords: Ischemia/reperfusion injury; Angiotensin II type 1 receptor; Angiotensin II type 2 receptor; Anti-apoptosis; Valsartan

1. Introduction

Angiotensin (Ang) II is a potent vasoconstrictor peptide that

acts through the rennin-angiotensin system (RAS) and elicits a

wide range of physiological actions (Edling et al., 1995). There

are two major Ang II receptor subtypes, Ang II type 1 receptors

Abbreviations: Ang, angiotensin; ARB, angiotensin II type 1 receptor

blocker; AT1, angiotensin II type 1 receptor; AT2, angiotensin II type 2

receptor; Bcl-2, B-cell lymphoma protein-2; GFAP, glial fibrillary acidic

protein; Jak/Stat, janus kinase/signal transducer and activator of transcription;

Map-2, microtubule associated protein-2; MCA, middle cerebral artery;

MCAO, middle cerebral artery occlusion; NADPH, nicotinamide adenine

dinucleotide phosphate; NeuN, Neuron-Specific Nuclear Protein; PBS, phos-

phate-buffered saline; pStat3, phospho-signal transducer and activator of tran-

scription-3; RAS, rennin-angiotensin system; rCBF, regional cerebral blood

flow; Stat3, signal transducer and activator of transcription-3; TTC, 2,3,5-

triphenyltetrazolium chloride; TUNEL, terminal deoxynucleotidyl transferase-

mediated deoxyuridine triphosphate nick-end labeling.

* Corresponding author. Tel.: +81 3 3813 3111; fax: +81 3 5684 0476.

E-mail address: t_urabe@med.juntendo.ac.jp (T. Urabe).

0168-0102/$ – see front matter # 2008 Elsevier Ireland Ltd and the Japan Neuro

doi:10.1016/j.neures.2008.03.003

(AT1) and Ang II type 2 receptors (AT2). AT1 mediate most of

the central effects of Ang II on cardiovascular regulation

(increase in blood pressure), fluid balance (release of vasopres-

sin), and hormone secretion (dipsogenic response). Furthermore,

AT1 is the predominant form expressed in most regions of the

adult rat brain except the midbrain (Hohle et al., 1995).

Recent work revealed that Ang II also elicits significant

proinflammatory actions in the vascular wall, including the

production of reactive oxygen species (ROS), inflammatory

cytokines, and adhesion molecules (Sadoshima, 2000). These

effects are mediated, at least in part, via the cytoplasmic nuclear

factor-kB (NF-kB) transcription factor, to augment vascular

inflammation and induce endothelial dysfunction, thereby

stimulating atherogenesis. NF-kB regulates many cytokines

and chemokines including the janus kinase (Jak)/signal

transducer and activator of transcription-3 (Stat3) pathway

(Brasier et al., 2002).

The neuroprotective mechanism of AT1 blockade has been

extensively investigated. Walther et al. (2002) observed a

smaller infarct area after occlusion of the middle cerebral artery

science Society. All rights reserved.

N. Miyamoto et al. / Neuroscience Research 61 (2008) 249–256250

(MCA) in AT1-deficient mice. Moreover, Iwai et al. (2004)

demonstrated that valsartan, an AT1 blocker (ARB), reduced

the ischemic area after MCA occlusion in wild-type mice.

Furthermore, AT1 and AT2 are upregulated in the ischemic

penumbra (Li et al., 2005). These results suggest a role for AT1

stimulation in the development of brain ischemic lesions, and

that blocking this activity decreases ischemia by lowering

blood pressure and possibly via other mechanisms. Moreover,

there is little or no information about the time course of AT1

and AT2 expression and action, or the relationship between

ARB and Jak/Stat signaling in ischemic brain damage.

The present study was designed to determine the serial

changes and intracellular signaling cascade for AT1 and AT2. For

this purpose, studies were conducted using a mouse transient

focal ischemia model treated with valsartan at a dose that did not

cause hypotension or change cerebral blood flow (CBF).

2. Materials and methods

2.1. Experimental protocol

All animal procedures were approved by the Animal Care Committee of

Juntendo University. Male C57BL/6 mice (8-week-old, weight 20–22 g) were

obtained from Charles River Institute and maintained on a 12/12-h light/dark

cycle with continuous access to food and water. Mice were divided at random

into three groups. (1) Valsartan-treated animals (n = 64) received intraperitoneal

injections of valsartan (Novartis Pharma) at 3 mg/kg body weight every day for

7 days prior to the operation. This treatment schedule and dosage were based on

the pharmacokinetic profile of valsartan supplied by the manufacturer and on

our own preliminary experiments. (2) A control saline group (n = 64) received

intraperitoneal infusions of saline at a volume similar to that used in the

valsartan group. (3) Control sham-operated vehicle and valsartan-treated

groups. These mice (n = 8 for each subgroup) underwent the same aforemen-

tioned protocol without middle cerebral artery occlusion (MCAO).

Ischemia was induced in all cases by the intraluminal vascular occlusion

method as described previously (Hara et al., 1996). Briefly, mice were initially

anesthetized with 4.0% isoflurane and maintained on 1.0–1.5% isoflurane in 70%

N2O and 30% O2 using a small-animal anesthesia system. The tip of the laser-

Doppler probe was fixed on the area selected for regional cerebral blood flow

(rCBF) monitoring, which corresponded to the territory of the occluded MCA.

The left MCA was occluded for 60 m and then released for reperfusion. During

this procedure, the body temperature was kept at 37.0 � 0.5 8C using a heating

pad (Unique Medical, Tokyo, Japan). Systolic blood pressure was assessed with a

noninvasive tail-cuff system (Softron BP-98A NIBP, Softron Inc., Tokyo) in

conscious mice. Regional CBF was measured by laser-Doppler flowmetry before,

during, and after MCAO, as well as before sacrifice. Neurological function was

assessed using a standard scoring system (Komine-Kobayashi et al., 2006): 0: no

defect; 1: failure to extend right forepaw; 2: circling to right; 3: falling to right; 4:

inability towalk spontaneously. At 24 and 72 h (n = 4, each) after reperfusion, 1.5-

mm-thick coronal sections from throughout the brain were stained with 2% 2,3,5-

triphenyltetrazolium chloride (TTC) to evaluate the infarct volume, as described

previously (Komine-Kobayashi et al., 2006).

2.2. Immunohistochemistry

Immunohistochemistry was performed on 20-mm-thick free-floating coronal

sections, prepared as described previously (Komine-Kobayashi et al., 2006). After

incubation in 3% H2O2 followed by blocking in 10% normal goat serum (Dako

Corporation, Carpentaria, CA) in phosphate-buffered saline (PBS), the sections

were incubated overnight at 4 8C with antibodies against anti-AT1 (1:50, Santa

Cruz Biotechnology, Santa Cruz, CA), anti-AT2 (1:50, Santa Cruz Biotechnol-

ogy), B-cell lymphoma protein-2 (Bcl-2) (1:40, Santa Cruz Biotechnology), Stat3

(1:300, Cell Signaling Technology, Beverly, MA), and phospho-Stat3 (pStat3)

(1:100, Cell Signaling Technology). The sections were subsequently incubated

with secondary antibodies (Vectastain; Vector Laboratories, Burlingame, CA),

and immunoreactivity visualized using the avidin–biotin complex method (Vec-

tastain) as described previously (Komine-Kobayashi et al., 2006).

2.3. Double immunofluorescence histochemistry

Double immunofluorescence staining was performed by simultaneous incu-

bation of the sections overnight at 4 8C with two of the following primary

antibodies: anti-Stat3 (dilution, 1:100), anti-pStat3 (dilution, 1:50), anti-Bcl-2

(dilution, 1:40), anti-AT1 (dilution, 1:50), anti-AT2 (dilution, 1:50), anti-NF-kB

(1:25, Cell Signaling Technology), anti-nitrotyrosine (1:100, Upstate Biotechnol-

ogy, Lake Placid, NY), anti-Map-2 (a neuron-specific marker, dilution, 1:100;

Bio-Rad Technologies, Hercules, CA), anti-NeuN (a neuronal marker, dilution

1:100; Chemicon International Inc., Temecula, CA), or anti-glial fibrillary acidic

protein (GFAP, which is specifically expressed in astrocytes, dilution, 1:500;

Dako). The antigen retrieval method was used in the immunostaining of NF-kB.

Briefly, this involved heating the sections in 10 mM sodium citrate buffer, pH 6.0,

until boiling, then maintaining them at a sub-boiling temperature for 5 min. The

slides were cooled for 30 min. Primary-antibody binding was detected by

simultaneous incubation with Cy3-conjugated secondary antibody (dilution,

1:500; Jackson Immunoresearch Laboratories, West Grove, PA) and fluorescein

isothiocyanate-conjugated secondary antibody (dilution, 1:500; Jackson Immu-

noresearch Laboratories) for 1 h at room temperature. The sections were washed

with PBS and mounted on microslide glass with Vectashield Mounting Medium

(Vector Laboratories). The sections were examined with a confocal laser scanning

microscope (Axiovert 100 M) using the LSM 510 system (Zeiss, Tokyo). All

images were obtained from individual optical sections. Data were minimally

processed to generate superimposed images using Laser sharp processing and

Photoshop software (Adobe System, San Jose, CA).

2.4. SDS-PAGE and immunoblotting

Mice of each group were decapitated at 24 or 72 h after reperfusion (n = 5 for

each group). Samples were taken from two regions of the brain: the ischemic

region comprising the cortex and striatum, and the control region encompassing

thesame area on the contralateral side. Thesamples were lysed in CelLytic reagent

(Sigma Chemical Co., St. Louis, MO) containing protease inhibitors (Calbiochem,

La Jolla, CA). Protein concentration in the lysates was determined using the BCA

protein assay kit (Pierce, Rockford, IL). Aliquots containing 30 mg of protein were

subjected to 10% SDS-PAGE. The protein bands were transferred to polyviny-

lidene fluoride membrane (Bio-Rad). The membranes were blocked with Block-

Ace (Dainichi-Seiyaku, Japan) and then incubated with primary antibodies as

follows: anti-pStat3 (dilution, 1:5,000), anti-Bcl-2 (dilution, 1:1,000), anti-AT1

(dilution, 1:1,000), anti-AT2 (dilution, 1:1,000), anti-NF-kB (dilution, 1:1,000),

or anti-a-tubulin (dilution, 1:2,000; Santa Cruz Biotechnology). After incubation

with the appropriate horseradish peroxidase-conjugated secondary antibody

(dilution, 1:20,000; Amersham Life Science, Buckinghamshire, UK) for 1 h at

roomtemperature, immunoreactive bandswerevisualized in the linear rangeusing

the enhanced chemiluminescence ECL Western blotting system (Amersham).

Equal protein loading was confirmed by immunoblotting for a-tubulin.

2.5. TUNEL

We detected in situ DNA fragmentation by terminal deoxynucleotidyl

transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)

staining using an ‘In Situ Cell Death Detection Kit, TMR red’ (Roche,

Mannheim, Germany) on the 20-mm-thick free-floating coronal sections were

used. After incubation in 0.1% sodium citrate in 0.1% PBS containing 0.1%

Triton X-100, the sections were incubated with the TUNEL reaction mixture for

60 min at 37 8C in the dark.

2.6. Cell count and statistical analysis

An investigator blinded to the experimental groups counted the number of

stained cells in three predefined areas (Fig. 1A, 0.25 mm2) of each stained

section using Axio-Vision software (Zeiss). Values are expressed as mean

� S.E.M. One-way ANOVA followed by post hoc Fisher protected least

Fig. 1. (A) Schematic representation of neuronal damage in mice brain after

reperfusion, delineated by loss of Map-2 staining. The shaded area represents the

infarct zone (cortex and sub-cortex). Three areas subjected to immunohistochem-

ical analysis are illustrated: Pe, peri-infarct area; Tr, transition area; Co, ischemic

core area. (B) Temporal changes in rCBF. pre; before operation, during; during

middle cerebral artery occlusion (MCAO). (C) Systolic blood pressure before,

N. Miyamoto et al. / Neuroscience Research 61 (2008) 249–256 251

significant difference test was used to determine the significance of differences

for various indexes among the different groups. A P value less than 0.05 denoted

the presence of statistical significance.

3. Results

3.1. Valsartan reduces infarct volume and neurological

deficit

There was no significant difference between vehicle and

treated groups in rCBF (Fig. 1B) and systolic blood pressure

(Fig. 1C) during the whole process of ischemia and reperfusion

with 3 mg/kg of valsartan.

After ischemia/reperfusion injury, a white-stained infarct

area and severe neurological deficit were observed only in the

operation group. In mice pretreated with valsartan, the infarct

volume was significantly smaller relative to the vehicle group at

24 and 72 h after reperfusion, and the reduction of infarct

volume was more prominent in the cortex (Fig. 1D and E). For

the neurological deficit score, valsartan significantly enhanced

functional recovery (as reflected by the neurological score),

compared with mice treated with the vehicle (Fig. 1F).

3.2. Temporal profile of AT1 and AT2 receptor expression

Next, we assessed the time course of receptor expression. In

the vehicle group, both AT1 and AT2 were significantly

upregulated at 24 h, and such overexpression reached peak

levels at 72 h with AT1 the dominant type in the peri-infarct

area and ischemic transition area compared with the sham-

operated group. Valsartan treatment significantly decreased the

expression level of AT1, while significantly increasing that of

AT2, compared with the vehicle group (Fig. 2A–C). AT1, AT2,

and NF-kB were clearly detected in the stroke lesion by

immunoblotting as bands of 41 kDa, 44 kDa, and 65 kDa,

respectively (Fig. 3B). In the vehicle group, the intensity of AT1

increased on the stroke side in a time-dependent manner

compared with the valsartan group. In contrast, the intensity of

AT2 increased in the valsartan group, and NF-kB was not

different between the vehicle and valsartan groups.

Double immunostaining showed co-expression of AT1 and

GFAP (astrocytes), as well as AT2 and NeuN (neuron marker),

in the ischemic transition area. Interestingly, NF-kB was co-

expressed in glia with AT1 in the vehicle controls, and in

neurons with AT2 in the valsartan-treated group (Fig. 3A).

3.3. Effects of valsartan on nitrotyrosine formation

Next, we examined the relationship between AT1-astrocyte-

NF-kB signaling and ROS, based on a previous report of AT1-

astrocyte-NF-kB signaling and NADPH oxidase-producing ROS

during, and after MCAO with reperfusion. (D) Coronal sections from ischemic

mice brain stained with TTC in valsartan group and vehicle group after 24 h

reperfusion. (E) Infarct volume was compared between the vehicle and valsartan

groups of cortex and sub-cortex area after 24 and 72 h veh; vehicle group, val;

valsartan group. (F) Neurological deficit scores in the vehicle and valsartan

groups. Data are mean� S.E.M., n = 5 each group. *P < 0.05 versus vehicle.

Fig. 2. (A) AT1 (a, c, e, g, i, and k) and AT2 (b, d, f, h, j, and l) immunostaining in the peri-infarct area (a, b, g, and h), transition area (c, d, i, and j) and ischemic core

area (e, f, k, and l) of representative vehicle (a–f) and valsartan-treated mice brains (g–l) at 72 h after reperfusion. Scale bar = 40 mm. (B) Number of AT1 receptor-

positive cells in the peri-infarct area (a), transition area (b) and ischemic core area (c). (C) Number of AT2-positive cells in the peri-infarct area (a), transition area (b)

and ischemic core area (c). Data are mean � S.E.M., n = 5 each group. **P < 0.001 versus vehicle.

N. Miyamoto et al. / Neuroscience Research 61 (2008) 249–256252

(Sadoshima, 2000). AT1 co-existed with nitrotyrosine in the

ischemic transition area of both groups (Fig. 4A), but few

nitrotyrosine-immunoreactive glia were detected in the corpus

callosum area of the sham-operated mice treated with the vehicle

or valsartan. In the vehicle group, nitrotyrosine-positive cells,

characterized by short and thin processes, were detected in the

transition area from 72 h after reperfusion. At 7 days after

reperfusion, these cells increased in number and showed stronger

staining intensity. In the valsartan group, few weakly stained cells

were detected. Double immunostaining for nitrotyrosine and

GFAP was used to determine the coexistence of both substances

in glial processes (Fig. 4B). The percentage of nitrotyrosine-

positive cells relative to GFAP-positive cells was significantly

reduced in the valsartan group compared with the vehicle group

at 72 h and 7 days after the ischemia/reperfusion injury (Fig. 4C).

3.4. Valsartan reduces apoptotic cell death

The AT2-neuron-NF-kB pathway is reported to be associated

with the Jak/Stat signaling pathway (Sadoshima, 2000). In

addition, we demonstrated previously that Jak/Stat signaling is

upregulated by Bcl-2, a well-known inhibitor of both apoptotic

and necrotic cell death (Komine-Kobayashi et al., 2006). We

therefore further investigated the relationship between the AT2-

neuron-NF-kB pathway and apoptosis. AT2 was found together

with pStat3 in the valsartan group (Fig. 5A), but not in vehicle-

treated controls. In contrast, AT1 did not coexist with pStat3 in

either group (data not shown). TUNEL staining revealed a

smaller number of apoptotic cells in the ischemic transition

area in the valsartan group compared with the vehicle group,

especially at 72 h after reperfusion (Fig. 5B and C).

3.5. Effect of valsartan on temporal profile of pStat3

production

The pStat3 immunoreactivity in neurons was more prominent

in the peri-infarct area and transition area of the valsartan group at

24 h after reperfusion than in the vehicle group, and was still

apparent at 72 h after reperfusion. However, the pStat3-positive

cells were rarely observed in the ischemic core area of the vehicle

Fig. 3. (A) Double immunofluorescence staining was performed for GFAP (red

[a]), NF-kB (red [d and j]), NeuN (red [g]), AT1 (green [b and e]), and AT2

(green [h and k]) in the ischemic transition area of the vehicle (a–i) and valsartan

groups (g–l) at 72 h after reperfusion. Scale bar = 10 mm. (B) Western blot

analysis. Samples were prepared from the brain at 24 and 72 h after reperfusion.

veh, vehicle group; val, valsartan group; C, contralateral lesion; S, stroke side.

Fig. 4. (A) Double immunofluorescent staining of AT1 (b)/nitrotyrosine (NT, a)

at 72 h in the vehicle group. Scale bar = 10 mm. (B) Double immunofluorescent

staining of nitrotyrosine and GFAP in the transition area at 72 h after reperfu-

sion (a–c for the vehicle group and d-f for the valsartan group). Scale

bar = 40 mm. (C) Percentage of nitrotyrosine-positive cells relative to GFAP-

positive cells. Data are mean � S.E.M., n = 5 each group. **P < 0.001 versus

vehicle.

N. Miyamoto et al. / Neuroscience Research 61 (2008) 249–256 253

and valsartan groups (Fig. 6A and B). Double immunofluores-

cence labeling to identify whether the pStat3-expressing cells

were neurons (NeuN) or astrocytes (GFAP) was then undertaken.

GFAP did not colocalize with pStat3, but pStat3-positive cells

were NeuN-positive in the transition area at 24 and 72 h after

reperfusion (Fig. 7A). Western blot analysis using pStat3

antibody showed a specific single band in the stroke side with a

molecular size of 92 kDa (Fig. 7B). The band intensity increased

in a time-dependent manner in both the valsartan and vehicle

groups (Fig. 7B). In the valsartan group, the intensity of the band

increased on the stroke side (P < 0.001) in a time-dependent

manner compared with the vehicle group (Fig. 7C).

3.6. Effect of valsartan on temporal profile of Bcl-2

production

At 24 h after reperfusion, the number of Bcl-2-positive

neurons increased in the vehicle group, but the increase was

higher in the peri-infarct area and transition area in the valsartan

group. However, there were few Bcl-2-positive cells in the

ischemic core area of the vehicle and valsartan groups (Fig. 6A

and B). Double immunofluorescence labeling was used to

identify the Bcl-2-expressing cells. GFAP did not colocalize

with Bcl-2, but Map2-positive cells were Bcl-2-positive in the

transition area (Fig. 7Ad-f). Immunoblots showed strong Bcl-2

immunoreactivity in the stroke lesion as a protein band of

26 kDa (Fig. 7B). In the valsartan group, the intensity of the

band increased (P < 0.001) on the stroke side in a time-

dependent manner compared with the vehicle group (Fig. 7C).

To assess the association between Bcl-2 and pStat3, we

colocalized Bcl-2 and pStart3 in neurons of the transition area

at 72 h after reperfusion (Fig. 7Ag-i).

4. Discussion

In the present study, we evaluated the effects of AT1 and AT2

in a mouse model of focal cerebral ischemia/reperfusion injury.

Our results suggest that AT2 reduces apoptosis by activating

Bcl-2 via Jak/Stat pathway while AT1 produces ROS.

The antiapoptotic effects of AT1 antagonism in this study

might have resulted from activation of AT2 in the brain. These

receptors are associated with neuronal differentiation, regen-

Fig. 5. (A) Double immunofluorescent staining of pStat3 (a)/AT2 (b) in the

transition area at 72 h in the valsartan group. Scale bar = 10 mm. (B) TUNEL

staining in the ischemic transition area of vehicle (a) and valsartan-treated

groups (b) at 72 h after reperfusion. Bar = 40 mm. (C) Number of apoptotic cells

in the ischemic transition area. Data are mean � S.E.M., n = 5 each group.**P < 0.001, versus vehicle.

Fig. 6. (A) pStat3 (a, c, e, g, i, and k) and Bcl-2 (b, d, f, h, j, and l) immunostaining i

core area (e, f, k, and l) of representative vehicle (a–f) and valsartan-treated mice b

pStat3/Bcl-2-positive cells in the peri-infarct area (a), transition area (b) and isc**P < 0.001, versus vehicle.

N. Miyamoto et al. / Neuroscience Research 61 (2008) 249–256254

eration, and tissue repair (Culman et al., 2001). In addition,

Kagiyama et al. (2003) demonstrated increased expression of

AT2 and levels of Ang II in the ipsilateral and contralateral

ventral cortices after transient MCAO. Thus, inhibition of AT1

seems to enhance the interaction of Ang II with AT2 and

thereby initiate neuronal regeneration in ischemic penumbra.

Expression of pStat3 and Bcl-2 was upregulated in the

valsartan-treated group in this study after reperfusion. This

indicated that valsartan exerts a neuroprotective role by

inhibiting apoptosis through stimulation of the Jak/Stat signaling

pathway with subsequent activation of Bcl-2. Experimental

evidence suggests that cerebral ischemic stress activates the Jak/

Stat pathway with pathological contribution from neurons and

glia (Justicia et al., 2000). Suzuki et al. (2001) elucidated the

chronological, topographical, and cellular alterations in pStat3

protein in a transient cerebral ischemia rat model. Activation of

pStat3 results in Stat3 dimers, which subsequently translocate to

the nucleus (Shuai, 2000) and regulate the transcription of target

proteins, such as Bcl-2, which is known to protect against both

apoptotic (Israels and Israels, 1999) and necrotic death (Kane

et al., 1993). Moreover, overexpression of Bcl-2 protects against

postischemic cerebral neuronal death (Ferrer and Planas, 2003).

To our knowledge, our study is the first to demonstrate an

antiapoptotic role for valsartan, and suggests that this effect is

mediated via activation of Bcl-2 through the Jak/Stat signaling

pathway.

AT2 receptor expression increases following injury and

during tissue remodeling (Matsubara, 1998). Stimulation of

AT2 in the cardiovascular system antagonizes AT1 activation,

including downstream reduction of oxidative stress (Wu et al.,

n the peri-infarct area (a, b, g, and h), transition area (c, d, i, and j) and ischemic

rains (g–l) at 72 h after reperfusion. Scale bar = 40 mm. (B) Numbers of Stat3/

hemic core area (c). Data are mean � S.E.M., n = 5 each group. *P < 0.05,

Fig. 7. (A) Double immunofluorescence staining was performed for pStat3

(red, a, g), Map-2 (red, d), NeuN (green, b), and Bcl-2 (green, e and h) in the

transition area of valsartan-treated mice at 72 h after reperfusion. Scale

bar = 10 mm. (B) Western blot analysis of Stat3/pStat3/Bcl-2. Samples were

prepared from brains at 24 and 72 h after reperfusion (veh, vehicle group; val,

valsartan group; C, contralateral lesion; S, stroke side). (C) Densitometric

analysis. Data are mean � S.E.M. and expressed relative to the respective a-

tubulin levels. *P < 0.05, **P < 0.001, compared with the respective vehicle

group.

N. Miyamoto et al. / Neuroscience Research 61 (2008) 249–256 255

2001; Okumura et al., 2005). Therefore, we speculate that

blockade of AT1 receptors in the present study resulted in

stimulation of AT2 with unbound Ang II, as reported previously

(Wu et al., 2001; Cosentino et al., 2005), mediating the

inhibitory action of ARB on oxidative stress and ischemic brain

damage.

It is tempting to assign a universal role for NF-kB in the

context of this study (such as ‘‘neuroprotective’’ or ‘‘neurode-

generative’’). However, conflicting reports exist in the literature

regarding NF-kB activity in response to brain injury, probably

reflecting the region- and disease-specific nature of such studies,

as well as model differences. A growing body of literature,

including the present report, points to the protective role for this

transcription factor in modulating the responses to neuronal

injury. This may occur through activation of antiapoptotic

genes or through other mechanisms such as the prevention of

excitotoxicity. Moreover, NF-kB acts to target genes for

cytokines, growth factors, adhesion molecules, immunorecep-

tors, acute phase proteins, transcriptional regulators, enzymes,

and antiapoptotic molecules such as the c-myc, c-IAP-1, c-IAP-

2, Bcl-xL, and Bcl-2 genes (Lee and Burckart, 1998). Such genes

have been shown specifically to confer neuronal resistance to

ischemia-induced damage (Mattson and Camandola, 2001). Our

results might therefore indicate a dual function for NF-kB

signaling, through AT1 for neurodegenerative effects, and

through AT2 for neuroprotection.

ARB does not seem to pass through the blood–brain barrier.

However, oral administration of ARB inhibited the effects of Ang

II injected into the paraventricular nucleus (Nishimura et al.,

2000a,b), suggesting that peripherally administered valsartan is

effective on the brain. Valsartan is widely used in patients with

hypertension and cardiovascular disease, and together, our

results further support this indication. Since such patients are at

higher risk of stroke, valsartan could prophylactically reduce

ischemic brain damage, infarct volume, and neurological deficit

once such complications occur.

Acknowledgments

This study was supported in part by a High Technology

Research Center grant and a grant-in-aid for exploratory

research from the Ministry of Education, Culture, Sports,

Science and Technology, Japan. Valsartan was a kind gift from

Novartis Pharma, AG (Basel, Switzerland).

References

Brasier, A.R., Recinos 3rd, A., Eledrisi, M.S., 2002. Vascular inflammation and

the renin-angiotensin system. Arterioscler. Thromb. Vasc. Biol. 22, 1257–

1266.

Cosentino, R., Savoia, C., De Paolis, P., Francia, P., Russo, A., Maffei, A.,

Venturelli, V., Schiavoni, M., Lembo, G., Volpe, M., 2005. Angiotensin II

type 2 receptors contribute to vascular responses in spontaneously hyper-

tensive rats treated with angiotensin II type 1 receptor antagonists. Am. J.

Hypertens. 18, 493–499.

Culman, J., Baulmann, J., Blume, A., Unger, T., 2001. The renin-angiotensin

system in the brain: an update. J. Renin Angiotensin Aldosterone Syst. 2,

96–102.

Edling, O., Gohlke, P., Paul, M., Rettig, R., Stauss, H.M., Steckelings, U.M.,

Unger, T., 1995. The renin-angiotensin system: physiology and pathophy-

siology: physiology of the renin-angiotensin system. In: Schachter, M.

(Ed.), ACE Inhibitors: Current Use and Future Prospects. Martin Dunitz,

London, pp. 3–41.

Ferrer, I., Planas, A.M., 2003. Signaling of cell death and cell survival following

focal cerebral ischemia: life and death struggle in the penumbra. J.

Neuropathol. Exp. Neurol. 62, 329–339.

Hara, H., Huang, P.L., Panahian, N., Fishman, M.C., Moskowitz, M.A., 1996.

Reduced brain edema and infarction volume in mice lacking the neuronal

isoform of nitric oxide synthase after transient MCA occlusion. J. Cereb.

Blood Flow Metab. 16, 605–611.

Hohle, S., Blume, A., Lebrun, C., Culman, J., Unger, T., 1995. Angiotensin

receptors in the brain. Pharmacol. Toxicol. 77, 306–315.

Israels, L.G., Israels, E.D., 1999. Apoptosis. Stem Cells 17, 306–313.

Iwai, M., Liu, H.W., Chen, R., Ide, A., Okamoto, S., Hata, R., Sakanaka, M.,

Shiuchi, T., Horiuchi, M., 2004. Possible inhibition of focal cerebral

ischemia by angiotensin II type 2 receptor stimulation. Circulation 110,

843–848.

N. Miyamoto et al. / Neuroscience Research 61 (2008) 249–256256

Justicia, C., Gabriel, C., Planas, A.M., 2000. Activation of the JAK/STAT

pathway following transient focal cerebral ischemia: signaling through Jak1

and Stat3 in astrocytes. Glia 30, 253–270.

Kagiyama, T., Kagiyama, S., Phillips, M.I., 2003. Expression of angiotensin

type 1 and 2 receptors in brain after transient middle cerebral artery

occlusion in rats. Regul. Pept. 110, 241–247.

Kane, D.J., Sarafian, T.A., Anton, R., Hahn, H., Gralla, E.B., Valentine, J.S.,

Ord, T., Bredesen, D.E., 1993. Bcl-2 inhibition of neural death:

decreased generation of reactive oxygen species. Science 262, 1274–

1277.

Komine-Kobayashi, M., Zhang, N., Liu, M., Tanaka, R., Hara, H., Osaka, A.,

Mochizuki, H., Mizuno, Y., Urabe, T., 2006. Neuroprotective effect of

recombinant human granulocyte colony-stimulating factor in transient focal

ischemia of mice. J. Cereb. Blood Flow Metab. 26, 402–413.

Lee, J.I., Burckart, G.J., 1998. Nuclear factor kappa B: important transcription

factor and therapeutic target. J. Clin. Pharmacol. 38, 981–993.

Li, J., Culman, J., Hortnagl, H., Zhao, Y., Gerova, N., Timm, M., Blume, A.,

Zimmermann, M., Seidel, K., Dirnagl, U., Unger, T., 2005. Angiotensin

AT2 receptor protects against cerebral ischemia-induced neuronal injury.

FASEB J. 19, 617–619.

Matsubara, H., 1998. Pathophysiological role of angiotensin II type 2 receptor

in cardiovascular and renal diseases. Circ. Res. 83, 1182–1192.

Mattson, M.P., Camandola, S., 2001. NF-kB in neuronal plasticity and neuro-

degenerative disorders. J. Clin. Invest. 107, 247–254.

Nishimura, Y., Ito, T., Saavedra, J.M., 2000a. Angiotensin II AT1 blockade

normalizes cerebrovascular autoregulation and reduces cerebral ischemia in

spontaneously hypertensive rats. Stroke 31, 2478–2486.

Nishimura, Y., Ito, T., Hoe, K., Saavedra, J.M., 2000b. Chronic peripheral

administration of the angiotensin II AT1 receptor antagonist candesartan

blocks brain AT1 receptor. Brain Res. 871, 29–38.

Okumura, M., Iwai, M., Ide, A., Mogi, M., Ito, M., Horiuchi, M., 2005. Sex

difference in vascular injury and vasoprotective effect of valsartan are

related to differential AT2 receptor expression. Hypertension 46, 577–583.

Sadoshima, J., 2000. Cytokine actions of angiotensin II. Circ. Res. 86, 1187–

1189.

Shuai, K., 2000. Modulation of STAT signaling by STAT interacting proteins.

Oncogene 19, 2638–2644.

Suzuki, S., Tanaka, K., Nogawa, S., Dembo, T., Kosakai, A., Fukuuchi, Y.,

2001. Phosphorylation of signal transducer and activator of transcription-3

(Stat3) after focal cerebral ischemia in rats. Exp. Neurol. 170, 63–71.

Walther, T., Olah, L., Harms, C., Maul, B., Bader, M., Hortnagl, H., Schultheiss,

H.P., Mies, G., 2002. Ischemic injury in experimental stroke depends on

angiotensin II. FASEB J. 16, 169–176.

Wu, L., Iwai, M., Nakagami, H., Li, Z., Chem, R., Suzuki, J., Gasparo, M.,

Horiuchi, M., 2001. Roles of angiotensin II type 2 receptor stimulation

associated with selective angiotensin II type 1 receptor blockade with

valsartan in the improvement of inflammation-induced vascular injury.

Circulation 104, 2716–2721.