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From the Department of Bioclimatology and Medicine, Medical Institute of
Bioregulation, Kyushu University, Beppu, Japan.
Correspondence to Naoki Makino, MD, Department of Bioclimatology and Medicine, Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumihara, Beppu, 874 Japan.
We used an antisense strategy to block circulating
angiotensinogen selectively. Antisense ODNs are widely used
as inhibitors of specific gene expression because they
offer an exciting possibility of blocking the expression of a
particular gene without any changes in the functions of other
genes.8 Therefore, antisense ODNs are considered
useful tools in the study of gene function and may also prove to be
potential therapeutic agents.9 However, antisense
ODNs still pose many unsolved problems, such as their short half-life,
low efficiency of uptake, and high degree of degradation by endocytosis
and nucleases.10 We recently developed an
intravenous gene transfer method mediated by the
asialoglycoprotein carrier molecule complex targeted to the
liver.11 This method can potentially be
successfully used to regulate liver gene
expression.12 13 14 15
The present study was therefore undertaken to determine the effect
of intravenous injection with antisense ODNs against
angiotensinogen to the liver on blood pressure and
angiotensinogen mRNA expression and plasma Ang II
concentrations in SHR. These antisense ODNs were originally designed to
be coupled to asialoglycoprotein carrier molecules, which
serve as important regulators of liver gene expression.
Construction of ODNs
Experimental Protocol
Biochemical Assay
Northern Blotting
Statistical Analysis
We also assessed the effect of antisense treatment on systolic
blood pressure (Fig 4
Regarding the antisense strategy to block circulating
angiotensinogen selectively, gene transfer methods have
recently been used as a delivery system of antisense ODNs in vivo as
described by others.5 6 7 Tomita et
al5 recently showed that an HVJ-liposome solution
containing ODNs against angiotensinogen transiently
decreased hypertension after administration via the hepatic portal
vein. They measured the blood pressure in the abdominal aorta through
an intra-arterial catheter. Under such conditions, the
original measurement of blood pressure in SHR was unreliable. In fact,
the blood pressure findings of Tomita et al were apparently lower than
our results. Although their methods, which were mediated by a viral
liposome complex, are efficient for gene transfer, many technical and
methodological difficulties still need to be overcome in comparison to
our method. In addition, such gene-targeting methods described above
are also troublesome to use in chronic clinical situations such as
treatment of hypertension. Wielbo et al7 also
showed that the peripheral administration of
liposome-encapsulated antisense via the carotid artery decreased the
blood pressure in SHR. They showed the change in mean
arterial pressure but not the original blood pressure of
the animals, whose blood pressure was assumed to be lower than that of
SHR. In addition, they did not show the effect of the
angiotensinogen mRNA expressions in vivo. In our study, the
intravenous injection used clearly revealed that the
reduction of the circulating angiotensinogen level resulted
in a decrease in the high blood pressure seen in the SHR for several
days. It is therefore expected to become clinically useful for the
treatment of hypertension in the future.
This study demonstrated the utility of gene transfer and antisense
technology for hypertension research, especially via the
intravenous injection of antisense ODN complexes. Although
we used the ODNs of phosphodiesters in the present study, our
antisense injection was considered to be successful for the following
reasons: the
asialoglycoprotein-poly(L)-lysine–antisense complex is
rapidly and preferentially taken up by the
liver12 13 14 15 and has enhanced resistance to
nuclease degradation in plasma.14 Within 10 to 20
minutes of intravenous administration of
asialoglycoprotein-poly(L)lysine–DNA complex, 80% to 85%
of the total amount is found in the liver, 80% of which is localized
specifically to hepatocytes.12 13
With biodistribution experiments using
32P-antisense, Lu et al15
showed that this conjugate was rapidly and preferentially taken up by
the liver with a concentration of
The intravenous injection of antisense ODNs results in a
significant decrease in blood pressure level from day 1 to day 5 after
injection compared with the sense ODNs treatment (P<.05).
The reduction in blood pressure was about 30 mm Hg, but the blood
pressure did not normalize. There fore, it may be assumed that blood
pressure regulation is affected not only by angiotensinogen
but also other diverse factors in vivo. Our results in blood pressure
did not correlate in percentage to the decrease in both plasma
angiotensinogen and Ang II levels. However, these
observations are remarkably similar to those recently published by
Gyurko et al19 20 who gave an antisense ODN to an
angiotensin type 1 receptor. On the other hand, because
angiotensinogen is one of the candidate genes for
hypertension from linkage genetic studies,2 3 our
findings also indicate that circulating angiotensinogen
plays an important role in the pathogenesis of hypertension of SHR. The
role of angiotensinogen in the regulation of blood pressure
was also previously demonstrated by the observation that the
administration of anti-angiotensinogen antibody resulted in
a reduction in blood pressure.21 In contrast, it
has also been reported that the acute administration of pure rat
angiotensinogen in rats caused an increase in blood
pressure.22
Our results did reveal that the reduction of the circulating
angiotensinogen level by antisense technology resulted in a
decrease in the high blood pressure seen in SHR, and these findings are
also consistent with those for angiotensinogen
knockout mice.23 It must, however, be mentioned
that our results were limited to a period of only 7 days after the
injection. Therefore, to elucidate the exact effect of
angiotensinogen on the development of hypertension and
hypertrophy, further long-term studies are called for.
Received November 14, 1997;
first decision November 24, 1997;
accepted December 31, 1997.
2.
Jeunemaitre X, Soubrier F, Kotelevetsev YV, Lifton RP,
Williams CS, Charru A, Hunt SC, Hopkins PN, Williams RR, Lalouel JM,
Corvol P. Molecular basis of human hypertension: role of
angiotensinogen. Cell. 1992;71:169–180.[Medline]
[Order article via Infotrieve]
3.
Caulfield M, Lavender P, Farrall M, Munroe P, Lawson
M, Turner P, Clark A. Linkage of the angiotensinogen gene
to essential hypertension. N Engl J Med. 1994;330:1629–1633.
4.
Ohkubo H, Kawakami H, Kakechi Y, Takumi T, Arai H,
Yokota Y, Iwai M, Tanabe Y, Masu M, Iwao H, Okamoto H, Yokoyama M,
Nomura T, Katsuki M, Nakanishi S. Generation of transgenic mice with
elevated blood pressure by introduction of the rat renin and
angiotensinogen genes. Proc Natl Acad Sci
U S A. 1990;87:5153–5157.
5.
Tomita N, Morishita R, Higaki J, Aoki M, Nakamura Y,
Mikami H, Fukamizu A, Murakami K, Kaneda Y, Ogihara T. Transient
decrease in high blood pressure by in vivo transfer of antisense
oligodeoxynucleotides against rat
angiotensinogen. Hypertension. 1995;26:131–136.
6.
Wielbo D, Serina C, Gyurko R, Phillips MI. Antisense
inhibition of hypertension in the spontaneously hypertensive rat.
Hypertension. 1995;25:314–319.
7.
Wielbo D, Simon A, Phillips MI, Toffolo S. Inhibition
of hypertension by peripheral administration of antisense
oligodeoxynucleotides. Hypertension. 1996;28:147–151.
8.
Helene C, Toulme JJ. Specific regulation of gene
expression by antisense, sense and antigene nucleic acids.
Biochem Biophys Acta. 1990;1049:99–125.[Medline]
[Order article via Infotrieve]
9.
Stein CA, Cheng Y-C. Antisense
oligonucleotides as therapeutic agents: is the bullet
really magical? Science. 1993;261:1004–1012.
10.
Agrawall S, Temsamani J, Tang JY. Pharmacokinetics,
biodistribution, and stability of oligodeoxy nucleotide
phosphorothioates in mice. Proc Natl Acad Sci U S A. 1991;88:7595–7599.
11.
Sugano M, Makino N. Changes in plasma lipoprotein
cholesterol levels by antisense
oligodeoxynucleotides against cholesterol ester
transfer protein in cholesterol-fed rabbits. J
Biol Chem. 1996;271:19080–19083.
12.
Wu GY, Wilson JM, Shalaby F, Grossman M, Shafritz DA,
Wu CH. Receptor-mediated gene delivery in vivo. J Biol
Chem. 1991;266:14338–14342.
13.
Chowdhury NR, Wu CH, Wu GY, Yerneni PC,
Bormmineni VR, Chowdhury JR. Fate of DNA targeted to the liver by
asialoglycoprotein receptor-mediated endocytosis in vivo.
J Biol Chem. 1993;268:11265–11271.
14.
Chiou HC, Tangco MV, Levine SM, Robertson D, Kormis K,
Wu CH, Wu GY. Enhanced resistance to nuclease degradation of nucleic
acids complexed to asialoglycoprotein-polylysine carriers.
Nucleic Acid Res. 1994;22:5439–5446.
15.
Lu XM, Fischman AJ, Jyawook SL, Hendricks K, Tompkins
RG, Yarmush ML. Antisense DNA delivery in vivo: liver targeting by
receptor-mediated uptake. J Nucl Med. 1994;35:269–275.
16.
Tokita Y, Franco-Saenz R, Reimann EM, Mulrow PJ.
Hypertension in the transgenic rat TGR (mRen-2) 27 may be due to
enhanced kinetics of the reaction between mouse renin and rat
angiotensinogen. Hypertension. 1994;23:422–427.
17.
Shimamoto K, Ishida H, Nakahashi Y, Nishitani T, Hosoda
S, Yokoyama T, Tanaka S, Iimura O. A very sensitive direct
radioimmunoassay system for plasma angiotensin II and its
clinical application in various hypertensive disease. Jpn Circ
J. 1984;48:1228–1235.[Medline]
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18.
Terada Y, Tomita K, Nonoguchi H, Marumo F. PCR
localization of angiotensin II receptor and
angiotensinogen mRNA in rat kidney. Kidney Int. 1993;43:1251–1259.[Medline]
[Order article via Infotrieve]
19.
Gyurko R, Tran D, Phillips MI. Time course of
inhibition of hypertension by antisense
oligonucleotides targeted to AT1
angiotensin receptor mRNA in spontaneously hypertensive
rats. Am J Hypertens. 1997;10:56S–62S.[Medline]
[Order article via Infotrieve]
20.
Gyurko R, Wielbo D, Phillips MI. Antisense
inhibition of AT1 receptor mRNA and angiotensinogen mRNA in
the brain of spontaneously hypertensive rats reduces hypertension of
neurogenic origin. Regul Pept. 1993;49:167–174.[Medline]
[Order article via Infotrieve]
21.
Gardes J, Bouhnik J, Corvol P, Menard J. Role of
angiotensinogen in blood pressure homeostasis.
Hypertension. 1982;4:185–189.
22.
Bouhnik J, Clauser E, Strosberg D, Frenoy JP, Menard J,
Corvol P. Rat angiotensinogen and des
(angiotensin I) angiotensinogen: purification,
characterization and partial sequencing. Biochemistry. 1981;20:7010–7015.[Medline]
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23.
Tanimoto K, Sugiyama F, Goto Y, Ishida J, Takimoto E,
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© 1998 American Heart Association, Inc.
Scientific Contributions
Intravenous Injection With Antisense Oligodeoxynucleotides Against Angiotensinogen Decreases Blood Pressure in Spontaneously Hypertensive Rats
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Abstract—In the
renin-angiotensin system, renin is known to cleave
angiotensinogen to generate angiotensin I,
which is the precursor of angiotensin II.
Angiotensin II is a vasoactive peptide that plays an
important role in blood pressure. On the other hand, the liver is the
major organ responsible for the production of
angiotensinogen in spontaneously hypertensive rats (SHR).
To test the hypothesis that a reduction of angiotensinogen
mRNA in the liver by antisense oligodeoxynucleotides (ODNs)
may affect both plasma angiotensinogen and
angiotensin II levels, as well as blood pressure, we
intravenously injected antisense ODNs against rat
angiotensinogen coupled to asialoglycoprotein
carrier molecules, which serve as an important regulator of liver gene
expression, into SHR via the tail vein. The SHR used in the present
study were studied at 20 weeks of age and were fed a standard diet
throughout the experiment. Plasma angiotensinogen,
angiotensin II concentrations, and blood pressure all
decreased from the next day until up to 5 days after the injection of
antisense ODNs. These concentrations thereafter returned to baseline by
7 days after injection. A reduction in the level of hepatic
angiotensinogen mRNA was also observed from the day after
injection until 5 days after injection with antisense ODNs. However, in
the SHR injected with sense ODNs, plasma angiotensinogen,
angiotensin II concentrations, and blood pressure, as well
as hepatic angiotensinogen mRNA, did not significantly
change throughout the experimental period. Although the exact role of
angiotensinogen in hypertension still remains to be
clarified, these findings showed that intravenous injection
with antisense ODNs against angiotensinogen coupled to
asialoglycoprotein carrier molecules targeted to the liver
could thus inhibit plasma angiotensinogen levels and, as a
result, induce a decrease in blood pressure in SHR.
Key Words: antisense elements • angiotensinogen • angiotensin II • blood pressure • genetics • rats, inbred SHR
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
The RAS acts to
constrict vessels and enhance the renal retention of sodium and water,
which thus induces an increase in blood pressure.
Angiotensinogen is synthesized in the liver and released
into the blood. It is cut by renin, which is produced by the kidneys
and then becomes Ang I.1 Ang I is cleaved by
angiotensin-converting enzyme into Ang II, which is an
active presser substance. Angiotensinogen has been
suggested to be an important determinant of both blood pressure and
electrolyte homeostasis.2 Recently, the potential
contribution of angiotensinogen in the pathogenesis of
hypertension has been suggested by genetic
approaches.2 3 The findings of some studies using
transgenic animals and a linkage analysis for the
angiotensinogen gene3 4 also support
this hypothesis. Regarding the role of circulating
angiotensinogen in the pathogenesis of hypertension, an
efficient gene transfer method mediated by a viral liposome complex has
been recently used as a delivery system of antisense ODNs in
vivo.5 6 7 However, many technical and
methodological difficulties still need to be overcome to use the
methods described above; the use of such gene targeting is also
troublesome in chronic clinical situations such as the treatment of
hypertension.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
All studies were performed with the approval of the Ethics
Committee on Animal Research of Kyushu University.
The sequences of ODNs against rat angiotensinogen
used in this study were as follows: antisense, 5'-CTGCTTACCTTTAGCT-3';
sense, 5'-AGCTAAAGGTAAGCAG-3'. These selected target sequences,
directed against the exon 1/intron 1 junction, have already been
described in the literature by Tomita et al5 and
inhibit the production of angiotensinogen. In
addition, they also have a relatively low homology with any of the
other known cDNA sequences found in the GenBank database. The synthetic
ODNs were purified on a reverse-phase high-performance liquid
chromatography column (Superdex FPLC,
Pharmacia Biotech), dried, resuspended in Tris-EDTA (10 mmol/L
Tris, 1 mmol/L EDTA, pH 7.4), and then quantified by
spectrophotometry.
Asialoglycoprotein-poly(L)lysine (approximate
molecular weight, [MW] 71.4 kD) prepared according to the method of
Wu (Wu et al12 and Chowdhury et
al13 ) was then added to the ODN (at a molar ratio
of 25:1) with vigorous mixing. The solution was incubated at 4°C
overnight and dialyzed (two times) against 0.15 mol/L saline (1500:1,
membrane MW cutoff, 3500). The samples were filtered through a 0.2-mm
membrane (Millipore Corp), electrophoresed through 2% agarose gel
using 0 Tris-borate-EDTA buffer, and then stained with ethidium bromide
to visualize DNA (Fig 1
). Sense or
antisense ODNs complex conjugated with the
asialoglycoprotein-poly(L)lysine was retained in the well,
whereas ODNs alone entered the gel. These complexes were then used for
the present study.
View larger version (45K):
[in a new window]
Figure 1. Asialoglycoprotein-poly(L)- lysine–ODN complex and
ODNs alone were electophoresed through 2% agarose gel using a
Tris-borate-EDTA buffer and then were stained with ethidium bromide to
visualize DNA. Lane 1,
Asialoglycoprotein-poly(L)lysine–ODN complex; lane 2, ODNs
alone. MM indicates Hae III molecular marker.
For the in vivo induction of ODNs in the present study, we
used 26 20-week-old SHR which were divided into two groups receiving
either antisense ODNs (n=13) or sense ODNs (n=13). All animals had ODNs
intravenously injected via the tail vein. Blood pressure
was measured everyday for 7 days by the standard tail-cuff method;
plasma Ang II concentration was then determined at 0, 3, 5, and 7 days
after ODN injection. On a predetermined day, the SHR were
anesthetized with pentobarbital (50 mg/kg IP), and liver tissue
specimens were removed to determine the angiotensinogen
mRNA expressions. All animals were housed at room temperature with
normal humidity and controlled light conditions. In addition, a
standard rat diet plus water was provided ad libitum.
After rats were killed by decapitation, blood was collected into
prechilled tubes containing EDTA (1 mg/mL whole blood). Plasma was
separated after being spun in a refrigerated centrifuge and was
stored at -80°C before analysis. For measurement of plasma
angiotensinogen concentration, 100 mL plasma was incubated
for 5 hours at 37°C with 5 mL 8-hydroxyquinoline, 5 mL dimercaprol,
25 mL Na2EDTA (4%), 50 mL rat kidney renin, and
65 mL Tris-acetate buffer (0.1 mol/L, pH 7.4) containing lysozyme; the
generated Ang I was measured by
radioimmunoassay.16 To measure the Ang II,
samples of freshly separated plasma were concentrated on an Amprep C8
minicolumn (Amersham International), and the Ang II content was
measured using very sensitive and specific anti–Ang II
antibody.17
Total RNA was isolated from the liver with a RNAzolB solution
(Biotex). The abundance of angiotensinogen mRNA was
determined with Northern blotting. The rat cRNA probe labeled with
fluorescein dUTP was produced by nonradiolabeled reverse
transcription polymerase chain reaction (PCR; Amersham) as described
previously11 according to the rat
angiotensinogen sequence.18 The sense
and antisense primers used for PCR and the size of PCR products in
each cRNA probe were as follows: angiotensinogen: sense
5'-TGGGGGT-TATCCAC-3', antisense 5'-CTGACCCAGTTCTTGCTGCC-3, size 482
bp; GAPDH: sense 5'-ATGGTCTACATGTTCCAGTA-3', antisense
5'-TAAGCAGTTGGTGGTGCAGG-3', size 343 bp. Poly(A+) RNA was isolated from
total RNA with Oligotex Super (Rosche), and the abundance of each mRNA
was determined by a Northern blot analysis.
All values are presented as the mean±SEM. The
statistical analysis was performed with a paired t
test for intragroup comparisons and Student's t test for
comparisons between the groups. Differences were considered to be
statistically significant at a value of P<.05.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Initially, to test whether the antisense strategy against
angiotensinogen inhibits the plasma
angiotensinogen concentration in SHR, we
intravenously injected antisense ODNs against
angiotensinogen coupled to asialoglycoprotein
carrier molecules from 10 to 50 µg into SHR via the tail vein (Fig 2B). At 3 days after the antisense ODN
injection, the plasma angiotensinogen levels significantly
decreased (P<.05) in a dose-dependent manner compared with
the preinjection levels (day 0). There was no difference in
angiotensinogen levels between 20 and 50 µg antisense ODN
(P>.05). When we injected 20 µg antisense ODN into SHR,
this level significantly decreased on days 3 and 5 after injection
(P<.05) compared with the sense ODN treatment (Fig 2A). In
contrast, the sense and antisense treatment groups did not differ
significantly on day 7 after injection (P>.05). We also
measured plasma Ang II levels in SHR after the injection of antisense
ODN complex (Fig 3). Ang II
concentrations were significantly decreased by antisense ODN injection
(P<.05). These results in Ang II concentration were similar
to the findings for angiotensinogen as shown in Fig 2.
View larger version (39K):
[in a new window]
Figure 2. Changes of the plasma angiotensinogen
concentrations in SHR after intravenous injection of
antisense ODNs against angiotensinogen coupled to
asialoglycoprotein carrier molecule. A,
Angiotensinogen concentration from day 0 to day 7 after
injection of 20 µg sense or antisense ODNs. B,
Angiotensinogen concentration on day 3 after the injection
of 0, 10, 20, or 50 µg antisense ODNs. Each bar indicates mean±SEM
of 5 samples. *P<.05 compared with data of day 0 or 0
µg; 
P<.05 compared with data for sense ODN
injection.
View larger version (36K):
[in a new window]
Figure 3. Changes of the plasma Ang II concentrations in SHR
after intravenous injection of antisense ODNs. A, Ang II
concentration from day 0 to day 7 after the injection of 20 µg sense
or antisense ODNs. B, Ang II concentration on day 3 after the injection
of 0, 10, 20, or 50 µg of antisense ODNs. Each bar indicates the
mean±SEM of 5 samples. *P<.05 compared with data of
day 0 or 0 µg; P<.05 compared with data for sense
ODN injection.). Systolic blood pressure began to
decrease on the next day (day 1) after injection. It then decreased
from 201±2.3 mm Hg on day 0 to 171±3.1 mm Hg on day 1
after the injection. The intravenous injection of antisense
ODNs resulted in a significant decrease in the blood pressure level
from day 1 to day 5 after injection compared with the sense ODN
treatment (P<.05), which is consistent with the
results for plasma Ang II levels. Next, to test the inhibition of
hepatic angiotensinogen production by
intravenous injection of antisense ODNs, we performed the
Northern blot analyses of hepatic angiotensinogen
mRNA treated with both antisense and sense ODNs. The hepatic
angiotensinogen mRNA levels did not differ significantly
between the sense-treated and untreated groups (data not shown). The
ratio of angiotensinogen mRNA to rat GAPDH mRNA also
significantly decreased on days 1, 3, and 5 after the injection of
antisense ODNs compared with the results on day 0 (Fig 5). There was no evidence that the
transfection itself changed the production of hepatic
angiotensinogen, which is an acute-phase protein, because
no significant change was observed in the hepatic
angiotensinogen content between untransfected and sense
ODN-transfected rats. On day 7, no change
was seen in the angiotensinogen mRNA expression between the
sense and antisense treatment groups after the injection. Moreover, the
results were not affected by hepatic dysfunction, as shown by the fact
that there was no liver toxicity. The liver function test results
showed no significant changes between the ODN-injected rats and the
untreated rats at 5 days after the injection (data not shown).
View larger version (15K):
[in a new window]
Figure 4. Changes of the systolic blood pressure in
SHR determined by the standard tail-cuff method from day 0 to day 7
after injection of sense ( 
) or antisense (
) ODNs. Each bar
indicates the mean±SEM of 7 to 13 experiments. *P<.05,
**P<.01, ***P<.001, compared with data
of day 0 as determined by a paired t test.
View larger version (33K):
[in a new window]
Figure 5. Representative Northern blot
analysis of hepatic angiotensinogen mRNA treated
with antisense ODNs from day 0 to day 7. GAPDH mRNA is indicated as the
control.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
In the present study, an injection of
asialoglycoprotein-poly-(L)lysine–antisense complex
reduced the plasma angiotensinogen levels and the hepatic
angiotensinogen mRNA as well as the systolic blood
pressure in SHR, whereas an injection of sense complex did not produce
a similar effect. The antisense ODNs used in the present study
demonstrated no side effects within 7 days after injection. Antisense
ODNs are widely used as inhibitors of specific gene
expression because they offer the possibility of blocking the
expression of a particular gene without inducing any changes in the
functions of other genes.8 9 These techniques may
give us new information about the pathobiology of hypertension. 
6% of the injected dose after
only 5 minutes and did not change significantly at 1 hour. In addition,
the total level of protein accumulation was significantly lower in
nonhepatic tissues. In the above time frame for intravenous
delivery, the vast majority of the ODNs of phosphodiesters bound to
asialoglycoprotein-poly(L)lysine conjugate remained
intact.14
Selected Abbreviations and Acronyms
Ang
=
angiotensin
ODN
=
oligodeoxynucleotide
RAS
=
renin-angiotensin system
SHR
=
spontaneously hypertensive rat(s)
Acknowledgments
This work was supported in part by a grant-in-aid from the
Ministry of Education, Science, and Culture, Japan. We wish to thank S.
Taguchi and M. Watanabe for their excellent technical
assistance.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
1.
Lynch KR, Peach MJ. Molecular biology of
angiotensinogen. Hypertension. 1991;17:263–269.
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