Temporary Pretreatment With the Angiotensin II Type 1
Receptor Blocker, Valsartan, Prevents Ischemic Brain
Damage Through an Increase in Capillary Density
Jian-Mei Li, MD, PhD; Masaki Mogi, MD, PhD; Jun Iwanami, MS; Li-Juan Min, MD, PhD;
Kana Tsukuda, BS; Akiko Sakata, MD; Teppei Fujita, MD;
Masaru Iwai, MD, PhD; Masatsugu Horiuchi, MD, PhD
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Background and Purpose—We investigated the effect of temporary treatment with a nonhypotensive dose of valsartan on
ischemic brain damage in C57BL/6 mice.
Methods—We separated the mice into 3 groups of valsartan treatment before middle cerebral artery (MCA) occlusion: (1)
for 4 weeks: Val (2W, 2W); (2) for 2 weeks followed by its cessation for 2 weeks: Val (2W, -); and (3) no treatment for
4 weeks: Val (-, -).
Results—Ischemic volume, DNA damage, superoxide production, and mRNA levels of monocyte chemoattractant
protein-1 and tumor necrosis factor-␣ on the ipsilateral side after 24 hours of MCA occlusion were significantly reduced
in both Val (2W, 2W) and Val (2W, -) mice compared with those in Val (-, -) mice, whereas these parameters were larger
in Val (2W, -) mice than in Val (2W, 2W) mice. Moreover, mice in both the Val (2W, 2W) and Val (2W, -) groups
exhibited an increase in cerebral blood flow in the peripheral territory of the MCA 1 hour after MCA occlusion, with
increases in endothelial nitric oxide synthase activation and nitric oxide production. Before MCA occlusion, treatment
with valsartan did not influence superoxide production or mRNA levels of monocyte chemoattractant protein-1 and
tumor necrosis factor-␣ in the brain. However, the capillary density in the brain in both Val (2W, 2W) and Val (2W, -)
mice was increased before MCA occlusion.
Conclusions—Our results suggest that temporary valsartan treatment could protect against ischemic brain damage even
after its cessation, at least in part due to an increase in capillary density. (Stroke. 2008;39:2029-2036.)
Key Words: angiotensin II type 1 receptor blocker 䡲 capillary density 䡲 cerebral blood flow
䡲 oxidative stress 䡲 stroke
A
ngiotensin (Ang) II is a potent vasoactive substance that
has a variety of physiological actions, including vasoconstriction, aldosterone release, and cell growth.1 Recent
clinical studies such as LIFE, MOSES, and the Jikei Heart
Study demonstrated that an Ang II receptor blocker (ARB)
prevented the onset of stroke beyond blood pressure-lowering
effects.2– 4 Consistent with clinical results, accumulating evidence from preclinical studies also indicates that ARB could
prevent the onset of stroke.5,6 Moreover, the possible involvement of the central renin–angiotensin system in ischemic
brain damage has been reported. Maeda et al reported that the
core lesion area after middle cerebral artery (MCA) occlusion was significantly reduced in angiotensinogen-knockout
mice.7 Walter et al and our group reported that a smaller
ischemic lesion area was observed after MCA occlusion in
Ang II type 1 (AT1) receptor-deficient mice.5,8 We previously
demonstrated that Ang II type 2 receptor null mice exhibited
a larger ischemic size and greater neurological deficit after
subjection to MCA occlusion.5 In addition, pretreatment with
an ARB has been reported to prevent ischemic brain damage
in various animal models.5,9 These results suggest that AT1
receptor stimulation is involved in the development of brain
ischemic damage, whereas Ang II type 2 receptor stimulation
could prevent it.
Despite the accumulating evidence, the detailed mechanisms of the protective effects of pretreatment with an ARB
on brain ischemic brain damage are still not well understood.
A recent clinical trial, TROPHY, showed that treatment of
prehypertension with an ARB, candesartan, reduced the
incidence of hypertension even after cessation of ARB
administration.10 This clinical study led us to examine the
possibility that temporary pretreatment with an ARB could
decrease brain ischemic damage after MCA occlusion in
mice. Moreover, we tried to explore the possible mechanisms
involved in this ARB’s potential protective effects on ischemic brain damage. Accordingly, we investigated the effects
Received September 5, 2007; final revision received November 12, 2007; accepted November 28, 2007.
From the Department of Molecular Cardiovascular Biology and Pharmacology, Ehime University Graduate School of Medicine, Tohon, Ehime, Japan.
Correspondence to Masatsugu Horiuchi, MD, PhD, FAHA Department of Molecular Cardiovascular Biology and Pharmacology, Ehime University
Graduate School of Medicine, Shitsukawa, Tohon, Ehime 791-0295, Japan. E-mail [email protected]
© 2008 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org
DOI: 10.1161/STROKEAHA.107.503458
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July 2008
of pretreatment with an ARB, valsartan, focusing on oxidative stress, inflammation, and vascular remodeling before and
after MCA occlusion in mice.
Materials and Methods
Adult male C57BL/6 wild-type mice (CLEA, Tokyo, Japan) from 6
weeks (weight, 20 g) were used in this study. The mice were housed
in a room in which lighting was controlled (12 hours on, 12 hours
off) and room temperature was kept at 25°C. The Animal Studies
Committee of Ehime University approved the experimental protocol.
Experimental Protocol
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Mice were given a standard diet (MF; Oriental Yeast Co Ltd, Tokyo,
Japan) and water ad libitum. An ARB, valsartan (provided by
Novartis Pharma AG), was administered through an intraperitoneally
implanted osmotic minipump (model 1002; Alza) at a dose of 3
mg/kg per day. This dose of valsartan is a nonhypotensive dose, as
previously reported.11 The mice were separated into 3 groups based
on the treatment with valsartan before MCA occlusion (1): valsartan
(3 mg/kg per day) treatment for 4 weeks: Val (2W, 2W); (2) valsartan
treatment for 2 weeks followed by cessation of its administration for
2 weeks: Val (2W, -); and (3) no treatment with valsartan for 4
weeks: Val (-, -).
Middle Cerebral Artery Occlusion
Focal cerebral ischemia was induced by occlusion of the MCA by an
intraluminal filament technique according to a method previously
described.7,12 Brain samples were obtained 24 hours after MCA occlusion, and coronal sections of 1-mm thickness were immediately stained
with 2% 2,3,5-triphenyltetrazolium chloride as previously described.12,13 Neurological deficit was evaluated 24 hours after MCA
occlusion by use of neurological scores developed by Huang et al.14
Evaluation of Capillary Density
We evaluated capillary density using a recently developed microangiographic technique.15 or immunofluorescence in the cerebral cortex of the periinfarcted zone before and after MCA occlusion.
Capillary density was evaluated in the cerebral cortex of the
periinfarcted zone before and after MCA occlusion. In the brain
without MCA occlusion (ie, “before” MCA occlusion), we have
evaluated the capillary density in a similar region of the brain. First,
bovine desiccate albumin–fluorescein isothiocyanate (SigmaAldrich, St Louis, Mo; 10 mg/mL) was injected through the tail vein
at 200 ␮L per mouse just before sampling the brain. The number of
vessels was analyzed with a Leica DMI 6000B microscope (Leica
Microsystems, Wetzlar, Bensheim, Germany) at ⫻100 magnification. Capillary density in a cortical area of 1 mm2 was calculated from
5 sections in each mouse. Approximately 100 to ⬇700 capillaries were
included in the area of 1 mm2. Second, brain sections were fixed with
10% formalin and permeabilized with 0.1% saponin before incubation
with goat polyclonal anti-PECAM-1 antibody (Santa Cruz Biotechnology, Inc, Santa Cruz, Calif) and goat polyclonal anti-Glut-1 antibody
(Santa Cruz Biotechnology, Inc). The secondary antibody was rabbit
antigoat antibody (Molecular Probes, Carlsbad, Calif). Cells were
counterstained with DAPI (Vectashield; Vector Laboratories, Inc.,
Burlingame, Calif). Images were viewed at ⫻10 magnification with an
Axioskop microscope (Carl Zeiss, Oberkochen, Germany) using image
analysis software (Atto Densitograph, Tokyo, Japan).
Laser-Doppler Flowmetry
Cerebral blood flow was determined in the territory of the MCA by
laser-Doppler flowmetry using a flexible fiberoptic extension to the
master probe (Omegaflo FLO-C1; Omegawave, Inc, Tokyo, Japan)
as previously described.5 Laser-Doppler flow measurements do not
quantify cerebral blood flow per gram of tissue; use of them at
precisely defined anatomic landmarks serves as a means of comparing cerebral blood flows in the same animal serially over time.16
Changes in cerebral blood flow after MCA occlusion were expressed
as the percentage of the baseline value of laser-Doppler flowmetry.
Detection of Superoxide Anion
Histological detection of superoxide anion was performed as described previously.17 In brief, frozen, enzymatically intact, 10-␮mthick sections were prepared from mouse brain 24 hours after MCA
occlusion and incubated immediately with dihydroethidium
(10 ␮mol/L) in phosphate-buffered saline for 30 minutes at 37°C in
a humidified chamber protected from light. Dihydroethidium is
oxidized on reaction with superoxide to ethidium, which binds to
DNA in the nucleus and fluoresces red. For detection of ethidium,
samples were examined with an Axioskop microscope (Axioskop 2
plus with AxioCam; Carl Zeiss) equipped with a computer-based
imaging system. The intensity of the fluorescence was analyzed and
quantified by means of computer imaging software (Densitograph;
ATTO Corp, Tokyo, Japan).
Real-Time Reverse Transcriptase–Polymerase
Chain Reaction
Real-time quantitative reverse transcriptase–polymerase chain reaction was performed with a SYBR kit (MJ Research, Inc, Waltham,
Mass). Polymerase chain reaction primers are shown in the supplemental Table I, available online at http://stroke.ahajournals.org.
Quantification of Damaged DNA
DNA damage was quantified with a DNA Damage Quantification
Kit (Dojindo, Kumamoto, Japan) based on the detection of abasic
sites in genomic DNA with an aldehyde reactive probe reagent that
reacts specifically with the open ring form of the abasic sites. We
performed this assay according to the manufacturer’s instructions.
Western Blot
Total protein was prepared from the brain, and Western blotting was
performed as previously described.18 Antibodies against phosphoendothelial nitric oxide synthase and total endothelial nitric oxide
synthase was purchased from Cell Signaling Technology, Inc (Danvers, Mass).
Detection of Nitric Oxide Production
Brain segments were loaded with 10 ␮mol/L diaminofluorescein-2
diacetate (Daiichi Pure Chemicals Co, Ltd, Tokyo, Japan) for 30
minutes at 37°C in HEPES buffer (pH 7.4). L-arginine (Wako Pure
Chemical Industries, Ltd, Osaka, Japan) at a dosage of 100 ␮mol/L
was put into the chamber during measurements to ensure adequate
substrate availability for nitric oxide synthase. The production of
nitric oxide was analyzed with a Leica DMI 6000B microscope at
⫻100 magnification.
Statistical Analysis
Values are expressed as mean⫾SEM in the text and figure. Data
were analyzed using analysis of variance followed by NewmanKeuls test for multiple comparisons. If a statistically significant
effect was found, post hoc analysis was performed to detect the
difference between the groups. Values of P⬍0.05 were considered
statistically significant.
Results
Focal Ischemic Injury of the Brain After Middle
Cerebral Artery Occlusion in Mice Treated
With Valsartan
Focal brain ischemia was induced by MCA occlusion with
the intraluminal filament technique. To examine the possible
protective effects of temporary treatment with valsartan on
brain ischemia, we separated C57BL/6J mice into 3 groups
(Val [2W, 2W], Val [2W, -], and Val [-, -]) as described in
“Materials and Methods.” As we previously reported,5 treatment with valsartan just before MCA occlusion (Val [2W,
2W]) decreased the ischemic area and its volume (Figure
Li et al
Effect of Temporal Pretreatment With ARB on Stroke
2031
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Figure 1. Ischemic area and volume, damaged DNA level, and neurological score 24 hours after MCA occlusion. A, 2,3,5-triphenyltetrazolium chloride staining of brain sections. Brain sections are numbered from frontal (section 1) to caudal (section 6). B, Morphometric
analysis of ischemic volume determined by 2,3,5-triphenyltetrazolium chloride staining and expressed as mm3. Comparison of (C) DNA
damage and (D) neurological scores. n⫽8 for each group. Val, valsartan (3 mg/kg per day). *P⬍0.05, **P⬍0.01 versus Val (-, -);
#
P⬍0.01 versus Val (-, -) ipsilateral; #P⬍0.05 versus Val (2W, -) Ipsilateral. Values are mean⫾SEM.
1A–B and supplemental Figure I, available online at http://
stroke.ahajournals.org). Interestingly, treatment with valsartan for 2 weeks followed by its cessation for 2 weeks (Val
[2W, -]) before MCA occlusion significantly decreased the
ischemic volume, although its protective effect seemed to be
less than that in Val (2W, 2W) mice (Figure 1B). We further
assessed the effects of valsartan on the DNA damage after
MCA occlusion by quantification of apurinic sites in genomic
DNA. We observed that MCA occlusion increased apurinic
sites on the ipsilateral side and that treatment with valsartan
for 4 weeks decreased apurinic sites on the ipsilateral side
(Val [2W, 2W]) with less but significant effects of treatment
with valsartan for 2 weeks (Val [2W, -]) even after its
cessation (Figure 1C). Treatment with valsartan did not
change apurinic sites before MCA occlusion (Figure 1C).
Neurological deficit after MCA occlusion evaluated by neurological score was also improved in both Val (2W, -) and Val
(2W, 2W) mice with more marked effects in Val (2W, 2W;
Figure 1D). Systolic blood pressure before and 24 hours after
MCA occlusion was not significantly different between these
3 groups.
Effects of Valsartan on Oxidative Stress and
Inflammatory Response in Mouse Brain Before
and after Middle Cerebral Artery Occlusion
To further assess the effects of temporary treatment with
valsartan on ischemic brain damage, we examined superoxide
anion production and inflammatory responses. Superoxide
anion production in the brain cortex before and 24 hours after
MCA occlusion was determined by dihydroethidium staining
(Figure 2A–B). We observed that superoxide anion production was enhanced on the ipsilateral side after MCA occlusion, and treatment with valsartan for 4 weeks before MCA
occlusion decreased this (Val [2W, 2W]) and temporary
treatment with valsartan also decreased superoxide anion
production (Val [2W, -]), whereas treatment with valsartan
did not influence superoxide production before MCA occlusion. Next, we examined mRNA expression of monocyte
chemoattractant protein-1 (MCP-1) and tumor necrosis
factor-␣ (TNF-␣) using real-time reverse transcriptase–polymerase chain reaction (Figure 2C–D). mRNA expression of
MCP-1 and TNF-␣ was increased on the ipsilateral side but
not on the contralateral side after MCA occlusion or before
MCA occlusion. Their expression in the ischemic area was
decreased in Val (2W, -) mice and further decreased in Val
(2W, 2W) mice.
Valsartan Increased Capillary Density and
Cerebral Blood Flow After Middle Cerebral
Artery Occlusion
Cerebral surface blood flow was measured in the core region
and peripheral region of the MCA territory. Cerebral blood
flow decreased just after MCA occlusion to approximately
10% of the basal level in the core region and to approximately
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50% in the periphery in Val (-, -) mice (Figure 3). This
reduction of cerebral blood flow continued for at least 24
hours after MCA occlusion. The decrease in cerebral blood
flow in the core was not significantly different between these
groups, whereas cerebral blood flow in the peripheral region
was significantly attenuated in Val (2W, -) and Val (2W, 2W)
mice (Figure 3). Cerebral blood flow in the peripheral region
was increased by valsartan treatment, but without a significant difference. To examine the possibility that administration of valsartan could increase vascular bed flow in response
Val (–, –)
Val (2W, –)
Val (2W,2W)
100
% Blood flow
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Figure 2. Superoxide anion production and inflammatory cytokines in the brain before and 24 hours after MCA occlusion A, Representative photographs of dihydroethidium staining in brain cortexes. B, Intensity analysis of superoxide production detected by dihydroethidium staining. Comparison of mRNA expression of (C) MCP-1 and (D) TNF-␣. n⫽8 for each group. Val, valsartan (3 mg/kg per day).
*P⬍0.05, **P⬍0.01 versus Val (-, -) before MCA; #P⬍0.01 versus Val (-, -) ipsilateral; #P⬍0.05 versus Val (2W, -) ipsilateral. Values are
mean⫾SEM.
75
**
*
*
50
25
0
Periphery **
Core
Before
0
1
24
Hours after MCA occlusion
Figure 3. Change in cerebral blood flow after MCA occlusion
Change in blood flow was expressed as a percentage of basal
flow. n⫽8 for each group. Val, valsartan (3 mg/kg per day).
*P⬍0.05 versus Val (2W, -) 0 hour after MCA occlusion;
**P⬍0.01 versus Val (2W, 2W) 0 hour after MCA occlusion. Values are mean⫾SEM.
to MCA occlusion, we examined capillary density. Using
confocal microscopy, capillary density in the brain was
calculated per surface area. Administration of valsartan for 4
weeks just before MCA occlusion (Val [2W, 2W]) increased
capillary density in the nonischemic and ischemic brain, as
shown in Figure 4A–C. Interestingly, an increase in capillary
density in the brain was also observed by temporary administration of valsartan (Val [2W, -]) before and after MCA
occlusion (Figure 4A–C).
Effect of Valsartan on Endothelial Nitric Oxide
Synthase and Nitric Oxide Production
To further explore the vasoprotective effects of valsartan in
terms of preventing ischemic brain damage, we examined the
possibilities that treatment with valsartan could increase
endothelial nitric oxide synthase (eNOS) expression, eNOS
activation, and nitric oxide production (Figure 5). Figure 5A
shows that serine phosphorylation of eNOS was induced on
the ipsilateral side but not on the contralateral side or before
MCA occlusion. This activation in the ischemic area was
enhanced by temporary administration of valsartan (Val [2W, -])
and further enhanced by administration of valsartan for 4 weeks
(Val [2W, 2W]). Consistent with the phosphorylation of eNOS,
nitric oxide production was also increased on the ipsilateral side
but not on the contralateral side or before MCA occlusion. This
increase in the ischemic area was enhanced in Val (2W, -) mice
and further enhanced in Val (2W, 2W) mice (Figure 5B–C).
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Figure 4. Comparison of capillary density in the cerebral cortex of the periinfarcted zone before and after MCA occlusion. A, Representative photos of the fluorescein isothiocyanate conjugated-labeled albumin stained capillaries in brain cortexes. B, Intensity analysis of
fluorescein isothiocyanate conjugated-labeled capillaries. C, Intensity analysis of platelet-endothelial cell adhesion molecule-1 (a) or
Glut-1 (b) -positive capillaries. Capillaries were expressed as capillaries per mm2. n⫽8 for each group. Val, valsartan (3 mg/kg per day).
*P⬍0.05, **P⬍0.01 versus Val (-, -) before MCA; #P⬍0.05, ##P⬍0.01 versus Val (-, -) ipsilateral. Values are mean⫾SEM.
Discussion
A recent clinical trial, TROPHY, showed that treatment of
prehypertension with an ARB, candesartan, reduced the
incidence of hypertension even after its cessation. In this
study, 2 years after cessation of candesartan, the incidence of
hypertension in the candesartan group remained lower than
that in the placebo group.10 Therefore, we examined the
possibility that temporary treatment with an ARB, valsartan,
could reduce ischemic brain damage after its cessation in a
mouse MCA occlusion model. In the present study, focal
cerebral ischemia was induced by a silicon-coated microfilament guided into the MCA. As we reported previously,5
administration of valsartan at a nonhypotensive dose attenuated the focal ischemic area induced by MCA occlusion.
Interestingly, temporary administration of valsartan significantly decreased the ischemic area, even after its cessation,
suggesting that valsartan could imprint some factors that
protect against ischemic brain damage.
The ischemic region can be separated into the infarct core,
in which oxygen supply is too low to sustain cell viability and
the ischemic penumbra.12 The penumbra is considered to be
a border zone between the region in which electric activity of
neurons stops and the region exhibiting membrane depolarization. In the present study, we measured surface cerebral
blood flow in the core and peripheral regions of the MCA
territory. The decrease in cerebral blood flow in the peripheral region (penumbra) was smaller in mice with continuous
treatment with valsartan before MCA occlusion (Val [2W,
2W]). Of note, temporary treatment with valsartan until 2
weeks before MCA occlusion increased cerebral blood flow
in the peripheral region. Treatment with valsartan could
increase the microcirculation, including formation of collaterals, thereby contributing to an increase in the vascular bed
and flow in response to MCA occlusion. To examine this
possibility, we examined the effects of valsartan on capillary
density in the brain before MCA occlusion. In the present
study, we observed that capillary density in the brain was
increased in valsartan-treated mice even before MCA occlusion and that even temporary treatment with valsartan increased capillary density. Recent studies indicate an inhibitory role of the AT1 receptor in capillary density. Li et al
reported that capillary density in the heart was reduced in
untreated C57BL/6 mice after nephrectomy; however, it was
significantly restored in both AT1 receptor null mice and
valsartan-treated mice.19 Their findings suggest that the
reduced myocardial capillary density is likely to be related to
augmented oxidative stress, which is thought to be mediated
by the AT1 receptor. Moreover, an AT1 receptor blocker,
losartan, has been shown to increase myocardial capillary
density in a rat model of myocardial infarction.20 Furthermore, Maxfield et al demonstrated that in streptozocin–
diabetic rats, endothelial capillary density, which was unaffected by diabetes, was increased by an AT1 receptor
antagonist.21 Xie et al also indicated that AT1 receptor
blockade by candesartan improved capillary density of the
left ventricular wall in stroke-prone spontaneously hypertensive rats.22 Interestingly, expression of vascular endothelial
growth factor and angiopoietin were increased by prostaglan-
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Figure 5. Expression of phosphorylated-eNOS and nitric oxide production in the brain before and 24 hours after MCA occlusion. A,
Western blot analysis of serine phosphorylation of eNOS. B, Representative photographs of the staining in brain cortexes. C, Intensity
analysis of nitric oxide production reacted with diaminofluorescein-2 diacetate. n⫽8 for each group. Val, valsartan (3 mg/kg per day).
*P⬍0.05, **P⬍0.01 versus Val (-, -) before MCA; #P⬍0.01 versus Val (-, -) ipsilateral; #P⬍0.05 versus Val (2W, -) ipsilateral. Values are
mean⫾SEM.
din F2␣-induced luteolysis in sheep to maintain vascular
structure, during which expression of the AT1 receptor was
decreased.23 Therefore, we speculate that valsartan pretreatment before MCA occlusion might have inhibited the AT1
receptor or/and increased vascular endothelial growth factor
and angiopoietin mRNA expression, resulting in an increase
in capillary density. In the recent clinical trial, the Jikei-Heart
study demonstrated that an addition of valsartan to conventional treatment reduced the onset of stroke to 40% less than
supplementary conventional treatment,3 indicating that treatment with valsartan has a beneficial effect on stroke onset
beyond an antihypertensive effect. Although preventive effects of ARBs on stroke have been discussed, our results also
suggest that hypertensive patients with ARB could be prevented from stroke partly through increasing capillary densities even after temporary treatment. Further studies to investigate the detailed mechanisms are needed in the future.
Oxidative stress is involved in various pathological processes,24 –26 and brain ischemia enhances oxidative stress.24,27
As reported previously, Ang II increases NADPH oxidase
activity and stimulates the production of reactive oxygen
species, including the superoxide anion, through the AT1
receptor.28 –30 MCA occlusion increased superoxide production in the ischemic area of the brain, and temporary treatment with valsartan (Val [2W, -]) attenuated superoxide
production, whereas continuous treatment with valsartan (Val
[2W, 2W]) decreased oxidative stress. Moreover, inflammatory responses are closely associated with ischemic brain
damage.31–33 The mRNA expression of MCP-1 and TNF-␣
was also increased in response to MCA occlusion, and
treatment with valsartan attenuated the inflammatory response assessed by MCP-1 and TNF-␣ mRNA expression.
However, treatment with valsartan (Val [2W, -], Val [2W,
2W]) did not apparently influence superoxide production or
mRNA expression of MCP-1 and TNF-␣ in the brain before
MCA occlusion. It was recently reported that in stroke-prone
spontaneously hypertensive rats, eNOS and nitric oxide
concentration were increased by valsartan, contributing to
vasodilation.34 Jiang et al also demonstrated that eNOS
expression and nitric oxide production were significantly
increased after hypoxia in endothelial progenitor cells.35 In
the present study, we observed that even temporary treatment
with valsartan increased activation of eNOS and nitric oxide
production on the ischemic side after MCA occlusion. We
speculate that the increase in capillary density and consequent
increase in cerebral blood flow by temporary treatment with
valsartan even after its cessation could attenuate oxidative
stress and inflammatory responses and increase nitric oxide
production. However, we did not observe such changes in
oxidative stress, inflammatory responses, and nitric oxide
production after the increases of capillary density and cerebral blood flow. In fact, as reported recently, the change in
superoxide production does not closely parallel the change in
capillary density. It has been demonstrated that in untreated
C57BL/6 mice after nephrectomy, capillary density in the
heart was reduced, whereas oxidative stress was markedly
increased, indicating that the change in oxidative stress does
not parallel the change in capillary density.19 Recent data
from D’Ascia et al demonstrated that myocardial tissue from
patients revealed a significant decrease in TNF-␣ expression
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with increased capillary density after 6 months of cardiac
resynchronization therapy.36 Moreover, endotoxemia tended
to decrease intestinal functional capillary density, whereas it
elevated TNF-␣ and interleukin levels in rats.37 It is suggested
that an increase in capillary density is not followed by an
increase in inflammatory responses. Furthermore, in a peritonitis model of sepsis in the rat, nitric oxide level increased
and contributed to decreased functional capillary density in
remote organs.38 Moreover, for evaluating the effects of ARB
on oxidative stress and inflammation before MCA occlusion,
it has to be taken in account that we used a nonhypotensive
mouse model in this experiment, which showed low basal
levels of oxidative stress and inflammation. Therefore, it is
possible that we cannot observe significant changes in superoxide anion production and MCP-1 and TNF-␣ expression by
valsartan treatment before MCA occlusion. In addition, it is
also possible that Ang II could exert diverse and biphasic
effects depending on Ang II concentrations, the levels of AT1
and Ang II type 2 receptors, and cell types in situ. Therefore,
to understand further the protective effects of pretreatment
with ARB on ischemic brain, more detailed analysis of Ang
II production and expression of Ang II receptors in situ have
to be evaluated.
In the present study, we demonstrated that temporary
treatment with an ARB, valsartan, for 2 weeks followed by its
cessation for 2 weeks attenuated brain damage after stroke.
This beneficial effect was induced partly due to increase in
capillary density before MCA occlusion; decrease in oxidative stress, inflammation, and DNA damage; and increase in
cerebral blood flow with an increase in nitric oxide production. In conclusion, these results provide new insights in the
protective effects of temporary ARB treatment on the ischemic brain even after cessation, at least in part due to
improvement of vascular remodeling, although further studies
are needed to explore the detailed mechanisms of the effects
of temporary treatment with valsartan concerning its protective effects in the ischemic brain.
Sources of Funding
This work was supported by grants from the Ministry of Education,
Science, Sports, and Culture of Japan (to M.H. and M.M.) and the
Suzuken Memorial Foundation (to M.M).
Disclosures
None.
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Temporary Pretreatment With the Angiotensin II Type 1 Receptor Blocker, Valsartan,
Prevents Ischemic Brain Damage Through an Increase in Capillary Density
Jian-Mei Li, Masaki Mogi, Jun Iwanami, Li-Juan Min, Kana Tsukuda, Akiko Sakata, Teppei
Fujita, Masaru Iwai and Masatsugu Horiuchi
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Stroke. 2008;39:2029-2036; originally published online April 24, 2008;
doi: 10.1161/STROKEAHA.107.503458
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