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Cardiovascular Research 1999 44(3):543-548; doi:10.1016/S0008-6363(99)00254-0
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Chronic antisense therapy for angiotensinogen on cardiac hypertrophy in spontaneously hypertensive rats

Naoki Makino*, Masahiro Sugano, Shoji Ohtsuka, Shojiro Sawada and Tomoji Hata

Departments of Bioclimatology and Medicine, Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumihara, 874-0838 Beppu, Japan

* Corresponding author. Tel.: +81-977-27-1676; fax: +81-977-27-1607

Received 14 April 1999; accepted 9 August 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
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 (n=18), and age-matched male Wistar–Kyoto rats (WKY, n=6) were used for this study. At 10 weeks of age, the SHR were divided into three groups (each group n=6), 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.

KEYWORDS Blood pressure; Gene expression; Gene therapy; Hypertrophy; Remodelling


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
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 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 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
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
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, 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 [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 (n=18) and age-matched male WKY rats (n=6) 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 (4°C) 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 –80°C 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 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 µg of total RNA was reverse transcribed into cDNA and then amplified using an RT-PCR kit (Gibco Life Technologies, Gaithersburg, MD, USA). The amplification profile involved denaturation at 95°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C 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 5'-ACCCCTTTCATCTCCTCTACT-3', the antisense primer was 5'-GGGTGTCTGGCTGCTGCTTCC-3', the PCR product size 488 bp, with 25 cycles. For ACE [23], the sense primer was 5'-CACCCTCTCGCTACAACTACG-3', the antisense primer was 5'-CCTCGCCATTCCGCTGATTCT-3', the PCR product size 408 bp, with 27 cycles. For AT1 receptor [24], the sense primer was 5'-GCCAAAGTCACCTGCATCAT-3', the anti-sense primer was 5'-AATTTTTTCCCCAGAAAGCC-3', the PCR product size 494 bp, and 27 cycles. For AT2 receptor [25], the sense primer was 5'-TGAGTCCGCATTTAACTGC-3', the anti-sense primer was 5'-ACCACTGAGCATATTTCTCAG-3', the PCR product size 436 bp, and 28 cycles. For glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) [26] as an inner control, the sense primer was 5'-GGTCTACATGTTCCAGTATG-3', the anti-sense primer was 5'-TAAGCAGTTGGTGGTGCAGG-3', 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(A+) 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 (RT-PCR) 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.

2.6 Statistical analysis
All data are given as the mean±S.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
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
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.


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  		Table 1 Left ventricular weight, left ventricle to body weight ratio, and systolic blood pressure in the experimental groupsa

 
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 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 (1.47±0.080) in WKY, (1.38±0.064) in the untreated SHR, (1.58±0.081) in the SHR injected with sense ODNs, and (0.68±0.035) 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.


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  		Table 2 Plasma angiotensinogen and angiotensin II levels and plasma angiotensin-converting enzyme activity in the experimental groupsa

 

Figure 1
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  		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.

 
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 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.


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  		Table 3 Hydroxyproline content, ACE activity, and the collagen volume fraction in the left ventricle in the experimental groupsa

 

Figure 2
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  		Fig. 2 Representative results of mRNA expressions for angiotensin-converting 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.

 

Figure 3
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  		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 ({square}), untreated SHR (diagonal shaded box) and SHR injected with sense (dotted box) or antisense ({blacksquare}) ODNs. The values are the mean±S.E.M. of six experiments. * P<0.05 vs. untreated SHR or SHR injected with sense ODN, {dagger} P<0.05 vs. SHR injected with antisense ODNs.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
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 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 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.

Time for primary review 30 days.


   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.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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