Continuous Activation of Renin-Angiotensin System Impairs
Cognitive Function in Renin/Angiotensinogen
Transgenic Mice
Shinji Inaba, Masaru Iwai, Megumi Furuno, Yumiko Tomono, Harumi Kanno, Izumi Senba,
Hideki Okayama, Masaki Mogi, Jitsuo Higaki, Masatsugu Horiuchi
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
Abstract—We examined the possibility that continuous activation of the human brain renin-angiotensin system causes
cognitive impairment, using human renin (hRN) and human angiotensinogen (hANG) gene chimeric transgenic (Tg)
mice. Cognitive function was evaluated by the shuttle avoidance test once a week from 10 to 20 weeks of age. The
avoidance rate in wild-type mice gradually increased. In contrast, the avoidance rate in chimeric hRN/hANG-Tg mice
also increased; however, no further increase in avoidance rate was observed from 14 weeks of age, and it decreased
thereafter. Cerebral surface blood flow was markedly reduced in 20-week-old hRN/hANG-Tg mice. Superoxide anion
production in the brain was already higher in 10-week-old hRN/hANG-Tg mice and further increased thereafter with
an increase in NADPH oxidase activity. Moreover, expression of p47phox and Nox4 in the brain of hRN/hANG-Tg mice
also increased. Administration of an angiotensin II type 1 receptor blocker, olmesartan (5.0 mg/kg per day), attenuated
the increase in blood pressure and ameliorated cognitive decline with enhancement of cerebral surface blood flow and
a reduction of oxidative stress in hRN/hANG-Tg mice. On the other hand, hydralazine (0.5 mg/kg per day) did not
improve the decrease in avoidance rate, and did not influence cerebral surface blood flow or oxidative stress in
hRN/hANG-Tg mice, in spite of a similar reduction of blood pressure to that by olmesartan. Moreover, we observed that
treatment with Tempol improved impaired cognitive function in hRN/hANG-Tg mice. These results suggest that
continuous activation of the brain renin-angiotensin system impairs cognitive function via stimulation of the angiotensin
II type 1 receptor with a decrease in cerebral surface blood flow and an increase in oxidative stress. (Hypertension.
2009;53[part 2]:00-00.)
Key Words: angiotensin II receptors 䡲 cognitive function 䡲 blood flow 䡲 oxidative stress 䡲 transgenic mice
D
ementia is a common serious health problem that
impairs quality of life. A continuous decline in cognitive
function occurs as the natural aging course in both humans
and animal models. Hypertension is a major risk factor for
cerebrovascular disease, including stroke, and contributes to
the development of vascular dementia.1 Several clinical
studies, such as Perindopril Protection Against Recurrent
Stroke Study, Systolic Hypertension in Europe, and Study on
Cognition and Prognosis in the Elderly, have shown that
antihypertensive drug treatment is associated with reduced
cognitive decline.2– 4 However, it is not still clear which
classes of antihypertensive drugs provide greater benefits
than others.
Activation of the renin-angiotensin system (RAS) plays
major roles in elevated blood pressure and the development
of cerebrovascular disorders. All of the components of the
classic RAS have been identified in the brain.5,6 Recent
clinical trials, such as the Losartan Intervention for Endpoint
Reduction in Hypertensive Study, Morbidity and Mortality
After Stroke, Eprosartan Compared With Nitrendipine for
Secondary Prevention, and the Jikei Heart Study, demonstrated that blockade of RAS with angiotensin (Ang) II
receptor blockers (ARBs) is effective to prevent the onset of
stroke, irrespective of their blood pressure–lowering effect.7–9
Moreover, we have reported previously that blockade of RAS
with ARBs attenuates ischemic brain damage.10,11 However,
the roles of Ang II in cognitive function are not well defined.
In addition, the relationship between continuous activation of
the brain RAS and cognitive function is not understood.
Therefore, in the present study, we tested the hypothesis that
continuous activation of the brain RAS is involved in impairment of cognitive function.
For this purpose, we used chimeric double transgenic (Tg)
mice of the human renin (hRN) and human angiotensinogen
(hANG) genes. The chimeric (hRN/hANG-Tg) mice were
produced by mating of hRN-Tg and hANG-Tg mice. These
Received September 24, 2008; first decision October 19, 2008; revision accepted November 6, 2008.
From the Departments of Molecular Cardiovascular Biology and Pharmacology (S.I., M.I., M.F., Y.T., H.K., I.S., M.M., M.H.) and Integrated Medicine
and Informatics (S.I., H.O., J.H.), Ehime University Graduate School of Medicine, Shitsukawa, Ehime, Japan.
Correspondence to Masatsugu Horiuchi, 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.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.108.123612
1
2
Hypertension
February 2009, Part II
*
120
100
80
60
40
20
0
90
Avoidance rate (%)
*
140
100
10 Weeks of Age
20 Weeks of Age
p<0.05
160
(mmHg)
Systolic blood pressure
180
80
*
70
*
50
40
*
Olm (–)
Hyd (–)
Olm (+)
Hyd (+)
*
*
30
*
*
WT
hRN/hANG-Tg
hRN/hANG-Tg: Olm
hRN/hANG-Tg: Hyd
20
10
WT
*
*
60
0
10
11
12
13
14
15
16
17
18
19
20
Age (Weeks)
hRN / hANG-Tg
Figure 1. SBP in hRN/hANG-Tg mice. SBP at 10 and 20 weeks
of age were measured as described in the Methods section.
Olmesartan (Olm; 5.0 mg/kg per day) and hydralazine (Hyd; 0.5
mg/kg per day) were administered orally from 4 weeks of age
throughout the experiment. n⫽5 for each group. *P⬍0.01 vs WT
mice and hRN/hANG-Tg mice treated with Olm or Hyd.
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
chimeric mice have been developed as a mouse model of
human hypertension induced by activation of the human
RAS.12–14 Our recent article showed that Ang II content is
significantly increased in the brain, as well as in plasma, in
hRN/hANG-Tg mice, and that activation of the human RAS
in the brain is involved in exaggeration of ischemic brain
damage attributed partly to enhancement of oxidative stress.15
Ang II is known to increase oxidative stress, and several
studies have demonstrated that impairment of cognitive
function is associated with an increase in oxidative stress.16 –18
Therefore, we examined the roles of continuous activation of
the human RAS in the brain, focusing on oxidative stress, as
well as cerebral blood flow.
Methods
Animals and Treatment
Adult male mice aged 10 weeks were used in this study. Transgenic
mice carrying both the human renin and angiotensinogen genes
(hRN/hANG-Tg) were generated by mating of human renintransgenic (hRN-Tg; C57BL/6J background) mice with human
angiotensinogen-transgenic (hANG-Tg; C57BL/6J background)
mice purchased from Riken Bioresource Center (Tsukuba, Japan).12
C57BL/6J mice were used as a genetic background–matched wildtype control. The animals were housed in a room with a 12-hour
light/dark cycle with a temperature of 25⫾1°C. They were given
standard laboratory chow (MF, Oriental Yeast Co, Ltd) and water ad
libitum. Olmesartan, a selective Ang II type 1 (AT1) receptor blocker
(5.0 mg/kg per day in laboratory chow, provided by Sankyo Co. Ltd),
and hydralazine (0.5 mg/kg per day in laboratory chow, Sigma-Aldrich Japan K.K.) were administered orally from 4 weeks of age
through the experiment (until a maximum of 20 weeks of age).
Tempol (1 mmol/L in drinking water, Sigma-Aldrich Japan K.K.), a
superoxide scavenger, was administered from 4 weeks of age
through the experiment (until a maximum of 20 weeks of age). The
Ehime University Animal Studies Committee approved the following experimental protocol. Blood pressure was measured by indirect
tail-cuff method with a blood pressure monitor (MK-1030, Muromachi Kikai Co, Ltd), as described previously.19 Mice were held in
a small plastic holder on a warming pad thermostatically controlled
at 37°C. Values were expressed as mean systolic blood pressure of
10 measurements in each group.
Evaluation of Cognitive Function by Shuttle
Avoidance Test
Cognitive function was evaluated by shuttle avoidance test (MBX002, Melquest).19,20 Briefly, a shuttle avoidance cage (32⫻12.5⫻15
Figure 2. Impairment of cognitive function in hRN/hANG-Tg
mice and its improvement by olmesartan. Shuttle avoidance test
using an electric shock was performed once a week from 10
weeks of age as described in the Methods section. n⫽5 to 6 for
each group. Olm indicates olmesartan; Hyd, hydralazine. E, WT
mice without treatment; F, hRN/hANG-Tg mice without treatment; f, hRN/hANG-Tg mice treated with Olm; 䡺, hRN/
hANG-Tg mice treated with Hyd. *P⬍0.05 vs WT and hRN/
hANG-Tg mice with Olm.
cm) was placed in an isolation cabinet (48⫻42⫻37 cm). The shuttle
avoidance cage was divided into 2 equal-size parts by a plastic
partition with a hole. The floor of the shuttle box consisted of
stainless-steel rods. An electric shock was delivered by a shock
generator to the stainless-steel rods on the floor of the cage. Each
mouse was placed in 1 side of the cage and given 20 trials. Each trial
consisted of a 5-second buzzer followed by an electric shock (0.3
mA, 3-second duration, 2-second interval) on the side of the mouse.
The side where the mouse was present was detected by a sensor.
When the mouse escaped the electric shock by moving from one side
to the other during the buzzer, it was counted as avoidance.
Avoidance rate was calculated as the ratio of avoidance in 20 trials.
The test was performed once a week between 10 AM and 12 PM.
Measurement of Cerebral Blood Flow
Cerebral surface blood flow (CBF) was determined under anesthesia
by laser speckle flowmetry (Omegazone laser speckle blood flow
imager, Omegawave, Inc), as described previously.21 Mean CBF was
measured in a region of the same size (250⫻300 pixels), where the
bregma was positioned at the center of the measured region. Images
were analyzed by the color image program incorporated in the
flowmetry system to obtain the average value of blood flow.
Detection of Superoxide Anion Production in
Brain Sections
Histological in situ detection of superoxide anion was performed
using freshly frozen sections stained with dihydroethidium
(10 ␮mol/L) in PBS for 30 minutes at 37°C, as described previously.11 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 using computerimaging software (Densitograph, ATTO Corporation).
Measurement of NADPH Oxidase Activity
Protein samples were obtained by homogenizing the brain with
ice-cold Tris-sucrose buffer (10 mmol/L of Tris [pH 7.0],
340 mmol/L of sucrose, and 1 mmol/L of EDTA). NADPH oxidase
activity was measured by cytochrome method and quantified from
the difference in absorbance with and without superoxide dismutase,
as described previously.22
Real-Time RT-PCR
Total RNA was extracted from the brain at 20 weeks of age.
Expression of mRNA was determined by quantitative real-time
RT-PCR. The level of target gene expression was normalized against
Inaba et al
A
Brain Angiotensin II System in Cognitive Function
A
10 Weeks of Age
10 Weeks of Age
20 Weeks of Age
20 Weeks of Age
WT
hRN/hANG-Tg
hRN/hANG-Tg
+ Olmesartan
hRN/hANG-Tg
+ Hydralazine
WT
hRN/hANG-Tg
hRN/hANG-Tg
+ Olmesartan
Stained with Dihydroethidium
50
10 Weeks of Age
20 Weeks of Age
p<0.05
40
*
*
30
20
10
0
WT
Olm (–)
Hyd (–)
Ol m (+)
300000
Intensity of fluorescense
(Arbitrary units)
p<0.05
B
Statistical Analysis
Values are expressed as means⫾SEMs in the text and figures. Data
were analyzed by 1-way ANOVA. If a statistically significant effect
was found, posthoc analysis was performed by Scheffe’s test to
detect the difference between the groups. A value of P⬍0.05 was
considered to indicate statistical significance.
Results
Systolic Blood Pressure in hRN/hANG-Tg Mice
Systolic blood pressure (SBP) in hRN/hANG-Tg mice was
higher than that in wild-type (WT) mice even at 10 weeks of
age and gradually increased thereafter, as reported previously.12,15 On the other hand, SBP in WT mice did not significantly
change during the experimental period (Figure 1).
Decrease in Cognitive Function in
hRN/hANG-Tg Mice
Cognitive function was evaluated by the shuttle avoidance
test. The avoidance rate was measured once a week from 10
*
100000
†
†
50000
WT
hRN / hANG-Tg
the GAPDH expression in each sample. PCR primers for p47
were 5⬘-GTCCCTGCATCCTATCTGGA-3⬘ (forward) and 5⬘-GGG
ACATCTCGTCCTCTTCA-3⬘ (reverse); for Nox4 they were 5⬘-GAG
TCACTCCATTTGCATCG-3⬘ (forward) and 5⬘-TCCCATCTGTTT
GACTGAGG-3⬘ (reverse); for the AT1 receptor they were 5⬘-AGT
CGCACTCAAGCCTGTCT-3⬘ (forward) and 5⬘-ACTGGTCCTTTG
GTCGTGAG-3⬘ (reverse); for the Ang II type 2 (AT2) receptor they
were 5⬘-CCTGCATGAGTGTCGATAGGT-3⬘ (forward) and 5⬘-CCA
GCAGACCACTGAGCATA-3⬘ (reverse); and for GAPDH were
5⬘-TGCGACTTCAACAGCAACTC-3⬘ (forward) and 5⬘-ATGTAG
GCCATGAGGTCCAC-3⬘ (reverse).
*
*
150000
0
phox
p<0.05
*
200000
Hyd (+)
Figure 3. Effect of olmesartan on CBF in hRN/hANG-Tg mice.
A, Representative photos. B, Quantitative analysis of CBF. n⫽5
to 6 for each group. Olm indicates olmesartan; Hyd, hydralazine.
*P⬍0.05 vs WT mice and hRN/hANG-Tg mice treated with Olm
at 20 weeks of age.
hRN/hANG-Tg
+ Hydralazine
10 Weeks of Age
20 Weeks of Age
p<0.05
250000
Olm (–)
Hyd (–)
Ol m (+)
Hyd (+)
hRN / hANG-Tg
C
NADPH oxidase activity
(pmol/µg/min)
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
Mean cerebral surface blood flow
(Arbitrary units)
B
3
10
*
8
*
*
*
†
6
†
4
2
0
WT
Olm (–)
Hyd (–)
Ol m (+)
Hyd (+)
hRN / hANG-Tg
Figure 4. Superoxide anion production and NADPH oxidase
activity in hRN/hANG-Tg mice. A, Representative staining of the
cerebral cortex with dihydroethidium. B, Intensity of fluorescence after dihydroethidium staining. C, NADPH oxidase activity
in the brain. n⫽5 to 6 for each group. Olm indicates olmesartan;
Hyd, hydralazine. *P⬍0.05 vs WT mice at the same age.
†P⬍0.05 vs hRN/hANG-Tg mice without Olm at the same age.
to 20 weeks. The avoidance rate in WT mice showed a
gradual increase. In contrast, the avoidance rate in hRN/
hANG-Tg mice also gradually increased; however, no further
increase in avoidance rate was observed from 14 weeks of
age, and it decreased thereafter (Figure 2).
Change in CBF in hRN/hANG-Tg Mice
CBF at the age of 10 weeks did not show a significant
difference between WT and hRN/hANG-Tg mice. However,
CBF in hRN/hANG-Tg mice markedly decreased, whereas
that in WT mice did not significantly change (Figure 3). At 20
weeks of age, CBF in hRN/hANG-Tg mice was ⬃80% of that
in WT mice.
4
Hypertension
February 2009, Part II
0.0020
0.0012
*
*
†
0.0008
0.0006
Nox4 mRNA
0.0010
0.0004
(Relative content to GAPDH)
p47 phox mRNA
(Relative content to GAPDH)
0.0014
*
*
0.0015
0.0010
†
0.0005
0.0002
0
WT
0
Olm (–) Olm (+) Hyd (+)
Hyd (–)
WT
Figure 5. Effect of olmesartan on expression of
p47phox (left) and Nox4 (right) mRNA in the
brain. Brain samples were taken at 20 weeks of
age. mRNA levels were measured by real-time
RT-PCR as the content relative to that of
GAPDH in the brain. n⫽5 to 7 for each group.
Olm indicates olmesartan; Hyd, hydralazine.
*P⬍0.05 vs WT mice. †P⬍0.05 vs hRN/
hANG-Tg mice without Olm.
Olm (–) Olm (+) Hyd (+)
Hyd (–)
hRN / hANG-Tg
hRN / hANG-Tg
Increase in Oxidative Stress in Brain of
hRN/hANG-Tg Mice
Treatment with an AT1 receptor blocker, olmesartan, from 4
weeks of age inhibited the increase in SBP to a similar level
compared with that in WT mice (Figure 1). Olmesartan
prevented impairment of cognitive function and reduction of
CBF in hRN/hANG-Tg mice throughout the experiment
(Figures 2 and 3). Moreover, olmesartan inhibited overproduction of superoxide anion and enhancement of NADPH
oxidase activity, together with suppression of expression of
NADPH oxidase subunits p47phox and Nox4 mRNA in the
brain (Figures 4 and 5). To examine the effect of elevated
blood pressure on cognitive function in hRN/hANG-Tg mice,
we next investigated the effect of treatment with hydralazine.
Treatment with hydralazine from 4 weeks of age reduced
0.0020
Treatment With Olmesartan Increased Expression
of AT2 Receptor mRNA in hRN/hANG-Tg Mice
There was no significant difference in the brain AT1 receptor
mRNA level in each group (Figure 6). AT2 receptor mRNA
level in the brain did not differ between WT and hRN/
hANG-Tg mice. Administration of olmesartan significantly
increased the AT2 receptor mRNA level, whereas treatment
with hydralazine did not influence it.
Effect of Antioxidant on Cognitive Decline in
hRN/hANG-Tg Mice
Effect of AT1 Receptor Blockade on Cognitive
Function in hRN/hANG-Tg Mice
Treatment with an antioxidant, Tempol, from 4 weeks of age
attenuated the impairment of cognitive function in hRN/
hANG-Tg mice (Figure 7A). In addition, administration of
Tempol improved the decrease in CBF (Figure 7B). SBP after
treatment with Tempol was slightly lower than that without
Tempol in hRN/hANG-Tg mice at the age of 20 weeks,
although there was no significant difference (119.8⫾6.5 mm Hg
in hRN/hANG-Tg mice with Tempol).
Discussion
Previous clinical studies have suggested that blockade of the
RAS could prevent cognitive impairment associated with
hypertension.2,4 Our results demonstrated that continuous
exaggeration of the human RAS in transgenic mice used in
0.0010
AT 2 receptor mRNA
0.0016
0.0012
0.0008
0.0004
0
WT
Olm (–) Olm (+) Hyd (+)
Hyd (–)
hRN / hANG-Tg
(Relative content to GAPDH)
AT 1 receptor mRNA
*
(Relative content to GAPDH)
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Superoxide anion production in the cerebral cortex detected
by dihydroethidium staining was already higher in hRN/
hANG-Tg mice than in WT mice even at 10 weeks of age and
increased thereafter (Figure 4A and 4B). We also observed
that NADPH oxidase activity in the brain was also enhanced
in hRN/hANG-Tg mice compared with that in WT mice
(Figure 4C). We next examined expression of NADPH
oxidase subunits p47phox and Nox4 mRNA in the brain at 20
weeks of age. Expression of p47phox (left) and Nox4 (right)
mRNA was significantly higher in hRN/hANG-Tg mice than
in WT mice (Figure 5).
SBP to a similar level to that caused by olmesartan but did not
improve the decrease in avoidance rate, CBF, or increase in
oxidative stress in hRN/hANG-Tg mice (Figures 1 to 5).
0.0008
Figure 6. Effect of olmesartan on expression of
AT1 (left) and AT2 (right) receptors in the brain.
Brain samples were taken at 20 weeks of age.
mRNA levels were measured by real-time
RT-PCR as the content relative to that of
GAPDH in the brain. n⫽5 to 7 for each group.
Olm indicates olmesartan; Hyd, hydralazine.
*P⬍0.05 vs WT mice and hRN/hANG-Tg mice
without Olm.
0.0006
0.0004
0.0002
0
WT
Olm (–) Olm (+) Hyd (+)
Hyd (–)
hRN / hANG-Tg
Inaba et al
90
80
Avoidance rate (%)
5
B
100
70
60
*
50
40
30
20
10
0
10 Weeks
of Age
15 Weeks
of Age
WT
20 Weeks
of Age
hRN/hANG-Tg
Mean cerebral surface blood flow
(Arbitrary units)
A
Brain Angiotensin II System in Cognitive Function
50
40
30
*
20
10
0
20 Weeks
of Age
Figure 7. Effect of antioxidant on cognitive function in hRN/hANG-Tg mice. Mice were treated with
an antioxidant, tempol, at 1 mmol/L in drinking
water from 4 weeks of age throughout the experiment as described in the Methods section. A,
Avoidance rate in hRN/hANG-Tg mice with or
without tempol. B, CBF in hRN/hANG-Tg mice
with or without tempol. Values of avoidance rate
and CBF in WT and hRN/hANG-Tg mice were
taken from Figure 2 and 3. n⫽5 to 6 for each
group. *P⬍0.05 vs WT mice and hRN/hANG-Tg
mice treated with tempol at 20 weeks of age.
hRN/hANG-Tg + Tempol
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
this experiment prevented the development of normal learning ability and, thereafter, impaired cognitive function. Moreover, administration of an ARB, olmesartan, ameliorated
cognitive decline, with a reduction of blood pressure. In
contrast, treatment with hydralazine did not improve cognitive decline, even with a similar decrease in blood pressure
compared with that by olmesartan. These results strongly
suggest that continuous exaggeration of RAS is involved in
impaired development of cognitive function. It is also reported that administration of Ang II or renin into the central
nervous system attenuates the retention of a passive avoidance task after learning23 and avoidance learning.24 The
shuttle avoidance test in our study is similar to a passive
avoidance test with electric shock to examine learning in
those previous articles. However, electric shock may cause
anxiety, which is difficult to be analyzed separately from
learning. Wilson et al25 reported that transgenic (mRen2)27
rats that overexpressed mouse renin with the increase in Ang
II showed an anxiatic profile, suggesting a possible involvement of Ang II in anxiety. Therefore, further examination is
needed to elucidate possible involvement of Ang II–induced
anxiety.
We demonstrated that continuous exaggeration of the
human RAS decreased CBF at 20 weeks of age. Overproduction of Ang II plays major roles in the pathogenesis of
cardiovascular and cerebrovascular diseases. Ang II induces
cerebrovascular remodeling, promotes vascular inflammation
and oxidative stress, and thereby impairs the regulation of
CBF.26,27 Previous studies showed that endothelial function in
cerebral vessels was impaired in a genetic model of Ang
II– dependent hypertension.13,28 It is known that CBF decreases with aging. Hypertension is a risk factor for cognitive
decline and dementia.1 Accumulating evidence suggests that
Alzheimer disease and other types of dementia, including
vascular dementia in humans, are associated with reduced
CBF.29 Moreover, it has been reported that hypertensive
patients had decreased CBF, although their cognitive function
was normal.30 These results indicate that reduction of CBF
could be involved in the development of neurodegenerative
disorders. In the present study, we demonstrated that the
decrease of CBF in hRN/hANG-Tg mice was improved by
the administration of olmesartan, but not hydralazine, although similar reductions of blood pressure were observed
with both drugs, supporting the notion that exaggeration of
AT1 receptor activation really plays a role in the impairment
of cognitive function. The reason that hydralazine did not
recover the decrease in CBF is not yet totally clear. However,
a possible explanation may be that the CBF in hRN/
hANG-Tg mice is mainly regulated by activated RAS in the
brain in this mouse model. In fact, as we have reported
previously, superoxide anion production was also markedly
increased in middle cerebral arteries of hRN/hANG-Tg mice,
which was inhibited by an ARB but not by hydralazine.15
Accordingly, some studies demonstrated that treatment with
an ARB exerted a protective effect on the autoregulation of CBF
in the spontaneously hypertensive rat via a reduction of
inflammation and superoxide generation, and restoration of
the vascular structure, independent of blood pressure.31,32
Moreover, we have reported previously that capillary density
in the brain was increased by treatment with an ARB.33 These
results suggest that these beneficial effects on cerebral vessels
by blockade of the AT1 receptor could contribute to suppression of the decrease in CBF.
In the present study, we demonstrated that superoxide
anion production, NADPH oxidase activity, and expression
of NADPH oxidase subunits, such as p47phox and Nox4, were
enhanced in the brain of hRN/hANG-Tg mice. It was reported
previously that the GAPDH mRNA level was decreased by
stimulation with Ang II in cultured vascular smooth muscle
cells.34 However, in the measurement of mRNA level, we
applied the same amount of total RNA, and the mRNA level
for GAPDH was not significantly different in all of the
experimental groups. Overproduction of Ang II induces
excessive release of superoxide radical by stimulation of
reduced nicotinamide-adenine dinucleotide/NADPH oxidase
via AT1 receptor activation. Yamamoto et al35 reported
previously that reactive oxygen species produced by Ang
II–activated NADPH oxidase increased cerebral neuronal
apoptosis and inflammation in stroke-prone spontaneously
hypertensive rats. Moreover, Wei et al27 reported that
NADPH oxidase contributed to vascular inflammation, endothelial dysfunction, and remodeling in transgenic (mRen2)27
rats, which exhibit elevated tissue Ang II, similar to our
experimental model, hRN/hANG-Tg mice. It has been recognized that oxidative stress is implicated in age-related
cognitive impairment.17,18 In addition, there are reports indi-
6
Hypertension
February 2009, Part II
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cating that oxidative stress is increased in the brain of patients
with Alzheimer disease and other neurodegenerative disorders.36 Experiments in Drosophila revealed that oxidative
stress was a causal factor in ␶-induced neurodegeneration.16
In the present study, administration of an antioxidant, Tempol, prevented the cognitive decline and the decrease in CBF
in hRN/hANG-Tg mice at 20 weeks of age. These results
suggest that the decline of cognitive function by continuous
activation of the brain RAS in hRN/hANG-Tg mice was
closely associated with enhanced oxidative stress because of
excessive stimulation of the AT1 receptor. There was no
significant difference in CBF at 10 weeks of age when the
oxidative stress was already elevated. This may be because of
a time difference between the elevation of oxidative stress
and its consequent effect on CBF. Therefore, we propose that
the sustained decrease in oxidative stress by blockade of the
AT1 receptor could contribute to neural protection and an
increase in CBF, resulting in the prevention of consequent
cognitive decline in hRN/hANG-Tg mice. Because it is
reported that Tempol reduces activity of the sympathetic
nervous system,37 there is a possibility that modulation of
sympathetic nerve activity is also involved in the actions of
Tempol and olmesartan.
Relative stimulation of AT2 signaling during ARB treatment has been highlighted in terms of protection against brain
damage.11,38 We observed that treatment with olmesartan
increased AT2 receptor expression in the brain. Therefore, we
can postulate that the effect of treatment with olmesartan to
prevent cognitive decline in hRN/hANG-Tg mice could
possibly be because of blockade of the AT1 receptor with
relative activation of the AT2 receptor. We have reported
previously that AT2 receptor signaling contributes to neuronal
differentiation and also that AT2 receptor signaling attenuates
DNA damage and vascular senescence.39,40 Therefore, the
inhibitory effect of treatment with olmesartan on cognitive
decline might be partly because of activation of the AT2
receptor in the brain of hRN/hANG-Tg mice. However, the
detailed mechanisms of the increase in expression of the AT2
receptor by olmesartan in the brain and its pathophysiological
relevance to prevention of cognitive decline need to be
addressed in more detail to further understand the roles of
exaggeration of RAS in cognition.
Perspectives
Our results suggest that continuous exaggeration of Ang II
production could result in impairment of normal development
of cognitive function and, consequently, cognitive decline,
probably independent of blood pressure increase. The decrease in CBF and continuous exaggeration of oxidative
stress induced by overproduction of Ang II could be involved
in cognitive decline. Taken together, our results provide new
insights into understanding how continuous long-term stimulation of the AT1 receptor could mediate cognitive impairment, and earlier treatment of hypertensive patients with an
ARB could have beneficial effects to prevent cognitive
decline.
Sources of Funding
This work was supported by grants from the Ministry of Education,
Science, Sports, and Culture of Japan.
Disclosures
None.
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Continuous Activation of Renin-Angiotensin System Impairs Cognitive Function in
Renin/Angiotensinogen Transgenic Mice
Shinji Inaba, Masaru Iwai, Megumi Furuno, Yumiko Tomono, Harumi Kanno, Izumi Senba,
Hideki Okayama, Masaki Mogi, Jitsuo Higaki and Masatsugu Horiuchi
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