Download Chronic antisense therapy for angiotensinogen on cardiac

Cardiovascular Research 44 (1999) 543–548
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Chronic antisense therapy for angiotensinogen on cardiac hypertrophy in
spontaneously hypertensive rats
Naoki Makino*, Masahiro Sugano, Shoji Ohtsuka, Shojiro Sawada, Tomoji Hata
Departments of Bioclimatology and Medicine, Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumihara, 874 -0838 Beppu, Japan
Received 14 April 1999; accepted 9 August 1999
Abstract
Objective: We examined the effect of the suppression of plasma angiotensinogen (AGT) by the intravenous injection of antisense
oligodeoxynucleotides (ODNs) against AGT targeted to the liver on cardiac remodeling in spontaneously hypertensive rats (SHR). The
ODNs against rat AGT were coupled to asialoglycoprotein (ASOR) carrier molecules, which serve as an important method for regulating
liver gene expression. Methods: Male SHR (n518), and age-matched male Wistar–Kyoto rats (WKY, n56) were used for this study. At
10 weeks of age, the SHR were divided into three groups (each group n56), and the systolic blood pressure (SBP) did not significantly
change among them. The control SHR and WKY groups received saline, the sense SHR group was injected with the sense ODNs complex
and the antisense SHR group was injected with the antisense ODNs complex, from 10 to 20 weeks of age. ASOR–poly( L)lysine–ODNs
complex was injected via the tail veins twice a week. Results: At the end of the treatment, a reduction of hepatic AGT mRNA, cardiac
angiotensin II type 1 receptor mRNA and the plasma AGT concentration was only observed in the antisense-injected SHR but not in the
other groups of SHR and WKY. This antisense therapy did not significantly change the mRNA expression for angiotensin converting
enzyme, angiotensin II type 2 receptor and AGT in the left ventricle (LV) among all three groups. Although the plasma angiotensin II
(Ang II) concentration significantly decreased to the level of WKY after the antisense therapy, the SBP, LV to body weight ratio and %
collagen volume fraction also showed a reduction, however, these findings were still larger than in the WKY than in either the
sense-injected SHR or control SHR. Conclusion: The plasma AGT is considered to play a role in the development of cardiac hypertrophy
in SHR, but it has not a complete effects on cardiac remodeling even if the plasma Ang II levels are inhibited because of an insufficient
suppression of hypertension.  1999 Elsevier Science B.V. All rights reserved.
Keywords: Blood pressure; Gene expression; Gene therapy; Hypertrophy; Remodelling
1. Introduction
Angiotensinogen (AGT) has been suggested to be an
important determinant of both blood pressure and electrolyte homeostasis [1]. Recently, the potential contribution
of AGT to the pathogenesis of hypertension has been
suggested based on genetic approaches [1–4]. AGT is
mainly synthesized in the liver and released into the blood.
It is cleaved by renin, which is produced by the kidneys,
and thereafter becomes angiotensin (Ang) I [5]. Ang I is
cleaved by ACE into Ang II, which is an active pressure
substance. Ang II is considered to act as a growth-promoting factor in the cardiovascular system [6], while it also
*Corresponding author. Tel.: 181-977-27-1676; fax: 181-977-271607.
increases collagen synthesis in the interstitium of the heart
[7,8]. Angiotensin-converting enzyme (ACE) inhibitors
can lower the blood pressure in spontaneously hypertensive rats (SHR) mainly by reducing the production of Ang
II [9,10]. Long term hypertension is reported to be
associated with cardiovascular remodeling, which consists
of cardiac hypertrophy and an increase in the extracellular
matrix (especially collagen) [11]. ACE inhibitors suppress
such myocyte hypertrophy in experimental models possibly through blood pressure-independent mechanisms
[9,12]. Recently, a selective Ang II receptor antagonist has
been developed which inhibits the renin–angiotensin system more specifically than ACE inhibitors [10,13]. Two
main Ang II receptor subtypes, AT1 and AT2, have been
Time for primary review 30 days.
0008-6363 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved.
PII: S0008-6363( 99 )00254-0
544
N. Makino et al. / Cardiovascular Research 44 (1999) 543 – 548
identified, and some other subtypes have also been described [14]. Ourselves and other investigators have also
shown the effects of the Ang II receptor antagonists on
cardiac remodeling [10,13].
However, the role of AGT in cardiac remodeling has yet
to be elucidated. In order to determine how the plasma
AGT levels effect cardiac remodeling in clinical situations,
the plasma AGT levels have to be reduced in the experimental models over a long period of time. We recently
showed that intravenous injection with antisense ODNs
against AGT coupled to asialoglycoprotein carrier molecules targeted to the liver is able to inhibit the plasma AGT
and Ang II [15], and as a result, induced a decrease in the
systolic blood pressure in spontaneously hypertensive rats
(SHR). The present study was therefore undertaken to
determine the effect of the suppression of plasma AGT on
cardiac remodeling in SHR.
2. Methods
All studies were performed with the approval of the
Ethics Committee on Animal Research of Kyushu University, Japan and conform with the Guide for the Care
and Use of Laboratory Animals published by the US
National Institutes of Health (NIH Publication No. 85-23,
revised 1996).
2.1. Construction of ODNs
The sequences of oligodeoxynucleotides (ODNs) against
rat angiotensinogen used in this study were as follows:
antisense, 59-CTGCTTACCTTTAGCT-39; sense, 59-AGCTAAAGGTAAGCAG-39. These selected target sequences, directed against the exon 1 / intron 1 junction,
have already been described in the literature [37] and
inhibit the production of angiotensinogen. The synthetic
ODNs were purified on the column, dried, resuspended in
Tris–EDTA (10 mM Tris, pH 7.4; 1 mM EDTA) and then
quantitated by spectrophotometry at 260 nm. The
asialoglycoprotein–poly( L)lysine–ODNs complex used in
this study was prepared as previously described [15,16].
2.2. Experimental protocol
Male SHR at 5 weeks of age (n518) and age-matched
male WKY rats (n56) were used for this study. All
animals were housed in a temperature, humidity, and
light-controlled room with free access to a standard rat diet
plus water. At 10 weeks of age, 18 SHR were divided to
three groups (six animals in each group); the systolic blood
pressure did not differ significantly between the control
and the experimental group. After 10 weeks, the control
group was injected with saline, the sense group was
injected with asialoglycoprotein (ASOR)–poly( L)lysine–
sense ODNs complex and the antisense group was injected
also with ASOR–poly( L)lysine–antisense ODNs complex
[17] and animals were kept for a further 10 weeks. Six
WKY rats were also injected with saline (0.2 ml) from 10
to 20 weeks of age (standard group). ASOR–poly( L)lysine–antisense ODNs complex was injected via the
tail veins twice a week. The amount of ODNs injected was
20 mg (0.2 ml) for each rat (i.e. 20 injections over 10
weeks). During the treatment period, both of body weight
and blood pressure (the standard tail-cuff method) were
measured once a week. At the end of treatment, the rats
were killed by decapitation and blood samples were drawn
into chilled tubes (48C) containing 0.1% EDTA to determined the plasma ACE activity. The left ventricle (LV)
was immediately removed, washed with ice-cold 10 mM
potassium phosphate buffer (pH 8.3), weighed and prepared to determine the tissue ACE activity, collagen
contents and the RNA isolation. The liver tissue specimens
were also removed and then were washed with ice-cold 10
mM potassium phosphate buffer (pH 8.3) to determine the
AGT mRNA expressions. The heart tissue specimens were
then frozen in liquid nitrogen and kept for up to 2 weeks at
2808C until the assays were performed. For the histological examination of the left ventricle (LV), the midportion
was fixed in 10% formalin for 2 days and embedded in
parafilm.
2.3. Biochemical assay
The plasma AGT and Ang II concentrations were
measured as described previously [10]. The left ventricular
collagen content was measured by the hydroxyproline
concentration of the tissue as described previously [18].
The ACE activity was measured by the modified method
of Hayakari et al. [19] as described previously [10].
2.4. Histological examination
The sliced sections were stained with hematoxylin–
eosin or Masson trichrome for fibrous regions. Ten sections per animal and ten fields per section were scanned
and computerized based on the staining levels. Transverse
diameter of cardiomyocytes was measured in LV free wall
according to the method previously described by Takemoto
et al. [20]. To determine the degree of collagen fiber
accumulation, we calculated the interstitial collagen volume fraction (%) as the sum of all connective tissue areas
of the entire visual field divided by the sum of all
connective tissue and muscle areas in the visual field of the
section with NIH image analysis software (NIH, Research
Service Branch) [21]. Perivascular matrix areas, as well as
artificial rupture areas, were excluded from this measurement.
2.5. Measurement of mRNAs
The total RNA was isolated from the LV tissues with a
N. Makino et al. / Cardiovascular Research 44 (1999) 543 – 548
545
Table 2
Plasma angiotensinogen and angiotensin II levels and plasma angiotensinconverting enzyme activity in the experimental groups a
RNAzolB solution (Biotex, Friendswood, TX, USA) according to the manufacturer’s procedure. ACE, AT1 and
AT2 mRNAs were measured by the reverse transcription
polymerase chain reaction (RT-PCR) as described previously [10] except for the fact that in the present study,
fluorescein 11-dUTP (Boehringer, Japan) was used to label
the PCR product. Briefly, 1 mg of total RNA was reverse
transcribed into cDNA and then amplified using an RTPCR kit (Gibco Life Technologies, Gaithersburg, MD,
USA). The amplification profile involved denaturation at
958C for 1 min, annealing at 588C for 1 min, and extension
at 728C for 1 min. We thereafter examined the relation
between the amount of RT-PCR products and the PCR
cycles (after determining the efficiency of amplification) in
each mRNA, the PCR cycles were next determined to
calculate the amount of each mRNA. For AGT [22], sense
primer was 59-ACCCCTTTCATCTCCTCTACT-39, the
antisense primer was 59-GGGTGTCTGGCTGCTGCTTCC-39, the PCR product size 488 bp, with 25 cycles. For
ACE [23], the sense primer was 59-CACCCTCTCGCTACAACTACG-39, the antisense primer was 59-CCTCGCCATTCCGCTGATTCT-39, the PCR product size 408
bp, with 27 cycles. For AT1 receptor [24], the sense primer
was 59-GCCAAAGTCACCTGCATCAT-39, the anti-sense
primer was 59-AATTTTTTCCCCAGAAAGCC-39, the
PCR product size 494 bp, and 27 cycles. For AT2 receptor
[25], the sense primer was 59-TGAGTCCGCATTTAACTGC-39, the anti-sense primer was 59-ACCACTGAGCATATTTCTCAG-39, the PCR product size 436 bp, and 28
cycles. For glyceraldehyde-3-phosphate-dehydrogenase
(GAPDH) [26] as an inner control, the sense primer was
59-GGTCTACATGTTCCAGTATG-39, the anti-sense
primer was 59-TAAGCAGTTGGTGGTGCAGG-39, the
PCR product size 343 bp, with 16 cycles. The abundance
of hepatic AGT mRNA was determined by a Northern blot
analysis using the DIG detection system (Boehringer,
Japan) after poly(A1) RNA was isolated from the total
RNA with Oligotex Super (Rosche, Japan). The rat cDNA
probes for AGT and GAPDH mRNA were produced by
the reverse transcription polymerase chain reaction (RTPCR) according to the rat sequence [22,26] as described
previously [15]. The cDNA probes were labeled with
digoxygenin-11-dUTP using DIG random prime labeling
system (Boehringer, Japan). The density of each PCR band
was analyzed with a densitometer (model 620, Japan Bio
Rad). The amount of mRNA was described as the ratio to
GAPDH.
AGT (pmol / ml)
Ang II (pmol / ml)
ACE activity
(nmol / min / ml)
WKY
SHR
Sense
Antisense
442623.9
21.663.1
44.263.4
456617.6
40.863.5*
91.764.9*
452619.4
39.164.3*
88.964.3*
267615.4* †
19.362.5 †
89.763.8*
a
Angiotensinogen, AGT; angiotensin II, Ang II; angiotensin-converting enzyme, ACE; * P,0.05 vs. WKY, † P,0.05 vs. SHR and sense. The
values are the mean6S.E.M. of six experiments.
2.6. Statistical analysis
All data are given as the mean6S.E.M. Comparisons
among three or more groups were made using a one-way
ANOVA followed by Dunnett’s modified t-test. The significance level P was set at 0.05.
3. Results
Table 1 shows the LV weight, the LV to body weight
ratio, and the systolic blood pressure in WKY and experimental SHRs at 20 weeks of age. Both of the LV
weight and the LV to body weight ratios were higher in the
20-week old SHR than in the same aged WKY. Both
parameters also significantly decreased in the SHR injected
with antisense ODNs compared to the untreated SHR or
SHR injected with sense ODNs, but not in comparison to
the levels of WKY. The systolic blood pressure in SHR
was significantly higher than that in the WKY. The
injection of antisense ODNs to SHR significantly reduced
the systolic blood pressure, which was still higher than that
of the WKY. The injection of sense ODNs had no effect on
the systolic blood pressure.
Table 2 shows the AGT, Ang II levels and ACE activity
in the plasma from experimental animals at 20 weeks of
age. The plasma AGT levels only decreased in the SHR
injected with antisense ODNs compared to WKY, the
untreated SHR and the SHR injected with sense ODNs.
The plasma Ang II levels in the SHR injected with
antisense ODNs significantly decreased to the level of the
WKY compared to the untreated SHR and the SHR
injected with sense ODNs. The plasma ACE activity in the
SHR significantly increased in comparison to the WKY but
was not significantly different between the untreated SHR
Table 1
Left ventricular weight, left ventricle to body weight ratio, and systolic blood pressure in the experimental groups a
LV weight (g)
LV/ body weight ratio
SBP (mmHg)
WKY
SHR
Sense
Antisense
0.9360.04
2.4860.05
14263.1
1.0860.06*
2.8760.06*
21966.1*
1.0660.04*
2.7860.07*
22164.8*
0.9560.03* †
2.6060.05* †
17864.6* †
a
Each value represents the mean6S.E. of six experiments. The sense or antisense ODNs for angiotensinogen was injected from 10 to 20-weeks old
SHR. LV, left ventricle; SBP, systolic blood pressure; * P,0.05 compared with WKY, † P,0.05 compared with the untreated SHR.
N. Makino et al. / Cardiovascular Research 44 (1999) 543 – 548
546
Fig. 1. Representative Northern blot analyses of angiotensinogen mRNA
in the liver from WKY, untreated SHR and SHR injected with sense or
antisense ODNs. GAPDH mRNA is used as an internal control.
and the SHR injected with sense ODNs or antisense
ODNs. Fig. 1 shows a typical example of Northern blot
analyses of hepatic mRNAs in each group. When the
amount of hepatic AGT mRNA was measured by scanning
and expressed as a ratio to GAPDH mRNA, the values
were (1.4760.080) in WKY, (1.3860.064) in the untreated
SHR, (1.5860.081) in the SHR injected with sense ODNs,
and (0.6860.035) in the SHR injected with antisense
ODNs. A significant reduction in the hepatic AGT mRNA
was only observed in the SHR injected with antisense
ODNs in comparison to the WKY, the untreated SHR and
the SHR injected with sense ODNs.
The ACE activity in the left ventricle of SHR also
significantly increased in comparison to the WKY (Table
3). This activity was not significantly different between the
untreated SHR and the SHR injected with sense ODNs or
antisense ODNs. The hydroxyproline concentrations, the
collagen volume fraction and the diameter of cardiac
myocytes all increased in the untreated SHR and the SHR
injected with sense ODNs compared with the WKY. These
values were significantly suppressed in SHR injected with
the antisense ODNs, but were still larger than in the WKY.
Fig. 2 shows a typical example of the RT-PCR products
for the animals in all four groups. The amount of AGT,
ACE, AT1 and AT2 mRNA levels in LV were measured by
scanning and expressed as a ratio to GAPDH mRNA, and
the findings are shown in Fig. 3. ACE, AT1 and AT2
mRNA increased more in the SHR than in the WKY but
they were not significantly different between the untreated
Fig. 2. Representative results of mRNA expressions for angiotensinconverting enzyme (ACE), angiotensin II type 1 receptor (AT1), angiotensin II type 2 receptor (AT2), and angiotensinogen (AGT) determined by RT-PCR methods in the left ventricle from WKY, untreated
SHR (SHR) and SHR injected with sense (sense) or antisense ODNs
(anti). GAPDH mRNA is used as an internal control.
SHR and the SHR injected with sense or antisense ODNs.
In addition, the AGT mRNA levels did not significantly
differ in any groups of SHR and WKY rats.
4. Discussion
The present study demonstrated that an injection of
ASOR–poly( L)lysine–antisense ODNs complex reduced
the hepatic AGT mRNA, the plasma AGT levels and the
plasma Ang II levels as well as the systolic blood pressure.
As a result, this complex suppressed the cardiac collagen
accumulation in the heart. The LV to BW ratio and the
diameter of cardiac myocytes significantly decreased in the
Table 3
Hydroxyproline content, ACE activity, and the collagen volume fraction in the left ventricle in the experimental groups a
Hydroxyproline
(mg / mg dry wt)
ACE activity
(nmol / min / mg protein)
Collagen volume
fraction (%)
a
WKY
SHR
Sense
Antisense
55.667.1
90.466.9*
88.667.4*
84.565.2*
2.7960.26
7.9060.64*
8.1160.59*
7.8260.49*
2.660.20
7.360.29*
6.860.38*
5.060.27* †
The values are the mean6S.E.M. of six experiments. * P,0.05 vs. WKY, † P,0.05 vs. SHR and sense.
N. Makino et al. / Cardiovascular Research 44 (1999) 543 – 548
Fig. 3. The amounts of mRNA levels for angiotensin-converting enzyme
(ACE), angiotensin II type 1 receptor (AT1), angiotensin II type 2
receptor (AT2), and angiotensinogen (AGT) were expressed as a ratio to
GAPDH mRNA in the left ventricle from WKY (h), untreated SHR
(diagonal shaded box) and SHR injected with sense (dotted box) or
antisense (j) ODNs. The values are the mean6S.E.M. of six experiments. * P,0.05 vs. untreated SHR or SHR injected with sense ODN,
†
P,0.05 vs. SHR injected with antisense ODNs.
SHR injected with antisense ODNs complex in comparison
to the untreated or sense injected SHR. However, those
results were more than the level of WKY. The
asialoglycoprotein carrier moiety used in the present study
was efficiently targeted to asialoglycoprotein receptor on
hepatocytes [17,27]. Although we did not evaluate either
the transfection efficacy or the stability of this antisense–
protein conjugate in the present study, Lu et al. [28]
demonstrated that the biodistribution pattern is consistent
with the mechanism for the specific uptake of the conjugate by the liver.
Although some genetic studies have reported on the
relationship between AGT and hypertension [1–3], the
findings remain controversial. Lodwiick et al. [29] reported
that the plasma AGT level did not differ between adult
SHR and WKY. In the present study, hepatic AGT mRNA
and the plasma AGT levels were not significantly different
between untreated SHR and WKY. The plasma AGT levels
were only reduced in the antisense injected SHR, while the
plasma Ang II levels apparently decreased in the antisense
injected SHR to the level of WKY. The plasma ACE
activity was higher in SHR than that of WKY, and did not
show to decrease in the antisense injected SHR as well as
the sense injected SHR in spite of the high amount of the
plasma AGT. AGT is cleaved by renin and thereafter
becomes Ang I [5], which is cleaved by ACE into Ang II.
Ang II affects both the blood pressure and cardiovascular
hypertrophy [6,8] and is therefore considered to act as a
growth-promoting factor directly on cardiac myocytes and
cardiac fibroblasts [7]. In the present study, the reduced
Ang II concentration in plasma is thought to be related to
the antihypertrophic action for cardiac myocytes in SHR.
In fact, ACE inhibitors, which inhibit Ang II production,
induce the regression of hypertrophy both in experimental
547
animals and humans either through blood pressure-dependent or independent mechanisms [9,12]. However, even
though our antisense injection reduced the plasma Ang II
to the level of WKY and partially suppressed cardiac
hypertrophy, it could not reduce the blood pressure to the
level of WKY. The antisense therapy also did induced a
reduction in the both of the diameter of cardiac myocytes
and the interstitial collagen accumulation although it still
remained well above the level in the WKY. The mRNA
expressions for ACE and AT2 in LV all increased in the
antisense injected SHR, and these findings were similar to
those for the sense injected SHR and the untreated SHR.
On the other hand, the AT1 mRNA expression decreased
in the antisense injected SHR, at this time the systolic
blood pressure also significantly suppressed by this antisense therapy although it was still higher than that of
WKY. SHR whose circulating angiotensinogen was reduced by antisense [30–32] or angiotensinogen-deficient
mice [33] could show a decreased blood pressure due to
lowered plasma Ang I and Ang II levels. The present study
has thus demonstrated for the first time that even if Ang II
is reduced by lowering AGT for a long period of time, it is
difficult to lower the blood pressure completely or to
suppress cardiac remodeling in SHR to the level of WKY
because of the high plasma ACE activity, the enhanced
tissue AGT, ACE, and AT2 mRNA expressions. If a
similar action such as that observed in SHR also exists in
human essential hypertension, then the strategy to lower
AGT and / or Ang II as a main effect should be reconsidered regarding the suppression of cardiac hypertrophy in
the clinical treatment of hypertension. AT1 receptor blockades have been shown to prevent the development of the
extracellular matrix [10,13,34] and collagen accumulation
to the same extent as ACE inhibitor [35,36]. Our recent
study also showed that the suppression of AT1 by ACE
inhibitor or AT1 antagonist played an important role in
reducing the collagen content in the heart of SHR [10].
The antihypertensive action of losartan is also believed to
be based on a blockade of AT1 receptors [13,34]. These
findings mean that Ang II taken up through AT1 receptor
in the heart thus pay an important role in both of the
myocyte hypertrophy and the collagen accumulation as
well as the regulation of blood pressure. It is important to
note that our results showed the effect of the plasma AGT
and / or Ang II on cardiac remodeling. Therefore, in order
to elucidate the effect of tissue AGT on cardiac remodeling, further studies are called for.
Acknowledgements
The authors thank Sachiyo Taguchi and Yoshikazu Itoh
for their valuable technical assistances. This work was
supported in part by a grant-in-aid from the Ministry of
Education, Science and Culture of Japan.
548
N. Makino et al. / Cardiovascular Research 44 (1999) 543 – 548
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