(Hypertension. 1995;26:131-136.)
© 1995 American Heart Association, Inc.
Articles |
Transient Decrease in High Blood Pressure by In Vivo Transfer of Antisense Oligodeoxynucleotides Against Rat Angiotensinogen
From the Department of Geriatric Medicine, Osaka (Japan) University Medical School (N.T., R.M., J.H., M.A., Y.N., H.M., T.O.); Institute for Molecular and Cellular Biology, Osaka University (Y.K.); and Department of Applied Biochemistry, University of Tsukuba, Ibaraki, Japan (A.F., K.M.).
Correspondence to Toshio Ogihara, MD, Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565, Japan.
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Abstract |
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Abstract The renin-angiotensin system plays an important role in blood pressure regulation. Angiotensinogen, which is mainly produced in the liver, is a unique component of the renin-angiotensin system, because angiotensinogen is only known as a substrate for angiotensin I generation. It is unclear whether circulating angiotensinogen is a rate-limiting step in blood pressure regulation. Recent findings of genetic studies and analyses suggest that the angiotensinogen gene may be a candidate as a determinant of hypertension. To test the hypothesis that angiotensinogen may modulate blood pressure, we transfected antisense oligonucleotides against rat angiotensinogen into the rat liver via the portal vein using liposomes that contain viral agglutinins to promote fusion with target cells, a technique that has been reported to be highly efficient. Transfection of antisense oligonucleotides resulted in a transient decrease in plasma angiotensinogen levels in spontaneously hypertensive rats from day 1 to day 7 after the injection, consistent with the reduction of hepatic angiotensinogen mRNA. Plasma angiotensin II concentration was also decreased in rats transfected with antisense oligonucleotides. Moreover, a transient decrease in blood pressure from day 1 to day 4 was observed, whereas transfection of sense and scrambled oligonucleotides did not result in any changes in plasma angiotensinogen level, blood pressure, or angiotensinogen mRNA level. Overall, our results demonstrate that transfection of antisense oligonucleotides against rat angiotensinogen resulted in a transient decrease in the high blood pressure of spontaneously hypertensive rats, accompanied by a decrease in angiotensinogen and angiotensin II levels. These findings suggest that angiotensin production may be directly proportional to circulating angiotensinogen concentration and also raise the possibility that angiotensinogen may be a potential target for antihypertensive drugs.
Key Words: oligonucleotides, antisense • parainfluenza virus type I • hypertension, genetic • rats, inbred SHR • gene therapy
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Introduction |
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Numerous studies have demonstrated that the renin-angiotensin system (RAS) plays an important role in blood pressure (BP) regulation and water electrolyte balance.1 2 In this system, renin cleaves angiotensinogen to generate angiotensin I (Ang I), which is the precursor of the vasoactive peptide Ang II. Angiotensinogen is therefore suggested to be an important determinant of BP and electrolyte homeostasis. Moreover, recent molecular biological findings led to the new concept that a tissue RAS may also play an important role in cardiovascular diseases such as hypertension.3 4 5 Recently, by genetic approaches, the potential contribution of angiotensinogen in the pathogenesis of hypertension has been suggested.6 7 8 Some reports using transgenic animals harboring rat and human angiotensinogen genes also supported this hypothesis.9 10 11 12 However, little is known about the role of circulating angiotensinogen in the pathogenesis of hypertension because of the lack of pharmacological drugs that selectively block angiotensinogen. The development of such agents may facilitate the understanding of the specific role of circulating angiotensinogen.
We use an antisense strategy to block circulating angiotensinogen selectively. Antisense oligodeoxynucleotides (ODNs) are widely used as inhibitors of specific gene expression because they offer the exciting possibility of blocking the expression of a particular gene without any changes in the functions of other genes.13 Therefore, antisense ODNs are useful tools in the study of gene function and may be potential therapeutic agents. However, antisense ODNs have many unsolved problems, such as their short half-life, low efficiency of uptake, and degradation by endocytosis and nucleases.14 15 16 Recently, we have developed an efficient gene transfer method mediated by a viral liposome complex.4 17 18 19 20 This delivery system also enhances the efficiency and prolongs the half-life of antisense ODNs in vitro and in vivo.21 22 In this study, we reasoned that circulating angiotensinogen concentration may be directly proportional to the BP level in spontaneously hypertensive rats (SHR). Here, we report that transfection of antisense ODNs against rat angiotensinogen resulted in a transient decrease in plasma angiotensinogen level and high BP in SHR. These data suggest that angiotensinogen is an important determinant in BP regulation.
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Methods |
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All studies were performed with the approval of the Ethics Committee of Animal Research, Osaka University Medical School.
Construction of ODNs
ODNs were synthesized on a 391 DNA synthesizer (Applied
Biosystems), purified by high-performance liquid
chromatography, washed with 70% ethanol, dried, and
resuspended in TE buffer (10 mmol/L Tris, pH 7.5, and 1 mmol/L EDTA, pH
8.0). Concentrations were determined with a spectrophotometer. The
following sequences were used: antisense rat
angiotensinogen 1: 5'-ACA-GCT-ATC-CCC-TGG-T-3'; antisense
rat angiotensinogen 2: 5'-CCC-AGA-CAA-GCA-CAG-C-3'; and
antisense rat angiotensinogen 3:
5'-CTG-CTT-ACC-TTT-AGC-T-3' (Fig 1).23 The
corresponding sense ODNs were used as controls. We also used scrambled
ODNs as a negative control (5'-AAG-ACA-TGA-CAG-GTA-G-3').
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Cell Culture
Five-week-old male Wistar rats were anesthetized with
pentobarbital (60 mg/kg IP) and used for preparation of isolated
hepatocytes by in situ perfusion of the liver with
Ca2+-free Hanks' solution and
collagenase.24 Hepatocytes were
resuspended at 1x106 cells per milliliter in
William's E medium (GIBCO), pH 7.4, supplemented with 10% fetal calf
serum. Cells in the dishes (60 mm in diameter) were incubated at 37°C
in a humidified atmosphere of 95% air and 5%
CO2.25
Preparation of Hemagglutinating Virus of Japan–Liposome
Solution
Hemagglutinating virus of Japan (HVJ; Sendai virus, Z strain)
was propagated in chorioallantoic fluid of embryonated eggs, as
previously described.17 18 Briefly, HVJ was collected by
centrifugation at 27 000g for 40 minutes
and suspended with balanced salt solution [BSS(-); 137 mmol/L NaCl,
5.4 mmol/L KCl, 10 mmol/L Tris-HCl, pH 7.6] overnight. This procedure
was repeated at least twice. The resuspended HVJ was stored at -4°C
and used within 1 week after purification. The hemagglutinating
activity of HVJ was determined as described
previously.17 18 One A540 (measured by OD 540
nm) of HVJ suspension contained 1 mg/mL protein and was equivalent to
15 000 hemagglutinating unit (HAU)/mL as an index of fusogenic
property. Lipids (phosphatidylcholine,
phosphatidylserine, and cholesterol)
were mixed at a ratio of 4.8:1:2 (wt/wt/wt) as described
previously.4 17 18 19 20 The lipid mixture (10 mg) in
tetrahydrofuran was deposited in a rotary evaporator. Antisense and
sense ODNs were incorporated into liposome by shaking and sonication.
The liposomes and HVJ, inactivated by UV irradiation (110 erg per
millimeter squared per second) for 3 minutes just before use, were
incubated at 4°C for 10 minutes and then at 37°C for 30 minutes
with gentle shaking (two strokes per second). This solution was
centrifuged by sucrose gradient. The top layer was collected
for use. In previous studies,17 18 we have used high
mobility group-1 (HMG-1) for gene transfer to enhance the migration of
plasmid DNA into the nuclei. However, in the antisense ODN transfer
shown in the present study, we did not use HMG-1 for ODN
transfection because ODNs can easily migrate into the nuclei without
HMG-1.
In Vitro Transfection of ODNs
Cells were washed with BSS, and HVJ-liposome solution with
different concentrations of ODNs was then added to the wells. The cells
were incubated at 4°C for 5 minutes and then at 37°C for 30
minutes. After medium was changed to fresh medium with 10% fetal calf
serum, the cells were incubated in a CO2 incubator.
In Vivo Introduction of HVJ-Liposome Solution
Eight-week-old male SHR were anesthetized with
pentobarbital (50 mg/kg IP). The abdomen was opened with a median
incision, and the liver and portal vein were exposed. A total volume of
2.5 mL final HVJ-liposome solution containing ODNs was injected into
the liver via the portal vein or by multiple direct injections.
Rat Angiotensinogen Assay
The angiotensinogen levels in each medium, tissue,
and plasma were determined indirectly by the measurement of Ang I
generated after incubation with excess recombinant human active renin,
as described previously.26 Samples were resuspended in
angiotensinogen radioimmunoassay buffer (150 mmol/L
Na2HPO4, 160 mmol/L NaCl, 3 mmol/L EDTA,
5% bovine serum albumin) and incubated with 1 Goldblatt unit
recombinant human active renin and 1 mmol/L phenylmethylsulfonyl
fluoride for 2 hours at 37°C.26 27 During the
incubation, angiotensinogen was completely converted to Ang
I.27 The Ang I level of each sample was determined by
radioimmunoassay, and the results are expressed as nanograms Ang I
generated per 106 cells.
Ang II Measurement
Ang II concentration in plasma was determined as described
previously.20 26 Samples of 1 mL freshly separated plasma
were promptly collected on an Amprep C8 minicolumn (Amersham
International), and their Ang II content was measured with a sensitive
anti–Ang II antibody and high-performance liquid
chromatography.20 26
BP Measurement
BP was measured by direct monitoring through an
intra-arterial catheter inserted into the abdominal aorta
via the right femoral artery of rats under ether
anesthesia.20 After the operation, BP was
recorded in conscious rats with a pressure transducer (model
TP-400T), monitors (models AT-601G and AT-641G), and a recorder
(model RTA-1300, all from Nihon Kohden Co).
RNA Analysis
The liver was promptly removed and immediately frozen in
liquid nitrogen and stored at -80°C before RNA extraction. Total RNA
was extracted from total liver with guanidine thiocyanate by
ultracentrifugation through a dense cushion of
CsCl.28 Poly(A)+ RNA was purified from the
total RNA on an oligo(dT) column.29 For Northern blot
analysis, 20 µg poly(A)+ RNA was subjected to
electrophoresis on a 1.5% agarose-formaldehyde denaturing gel and
transferred to a nitrocellulose membrane (Amersham). The filter was
baked, prehybridized, and hybridized to full-length cDNA for rat
angiotensinogen (provided by Dr Mori, Kyoto University) and rat GAPDH
probe (Amersham), both labeled with 32P. The filter then
was washed and exposed to x-ray film.
Statistical Analysis
Results are expressed as mean±SEM. Statistical analysis
was performed by ANOVA followed by multiple comparisons. Differences
were considered statistically significant at a value of
P<.05.
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Results |
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Initially, we evaluated three sets of antisense ODNs against rat angiotensinogen using primary rat hepatocytes to determine the effective sequences. As shown in Fig 2a, only rat angiotensinogen antisense ODN 3 had a significant inhibitory effect on the production and release of rat angiotensinogen into the medium (P<.01). However, rat angiotensinogen sense ODN 3 had no inhibitory effect on the production of rat angiotensinogen. There was no significant difference in rat angiotensinogen production/release into the medium between antisense 1 and 2 and sense 1 and 2. Therefore, we used antisense 3 and sense 3 ODNs as the effective antisense oligonucleotide sequences. Moreover, the findings that treatment with scrambled ODNs had no effect on angiotensinogen production/release into the medium (sense 3 ODNs, 18.1±0.6 ng Ang I/mL versus scrambled ODNs, 18.3±0.4 ng Ang I/mL, P=NS) confirmed the specificity of the effects of antisense ODNs. Dose-dependent effects of antisense ODNs were also examined. As shown in Fig 2b, transfection of antisense ODNs resulted in the inhibition of angiotensinogen production/release in a dose-dependent manner. Angiotensinogen levels in the medium of cells transfected with sense ODNs did not change significantly compared with the control group. Moreover, these effects were observed in a dose-dependent manner without toxicity (untransfected, 8.6±0.2x105 cells per well versus ODN-transfected, 8.5±0.4x105 cells per well, P=NS).
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indicates ratio of angiotensinogen level in the medium of
cells transfected with antisense oligodeoxynucleotides
to that of cells transfected with scrambled
oligodeoxynucleotides; 
, ratio of
angiotensinogen level in the medium of cells treated with
sense (S) oligodeoxynucleotides to that of cells treated
with scrambled oligodeoxynucleotides. n=8 per group.Next, we tested whether the antisense strategy against angiotensinogen is effective in vivo given the highly efficient gene transfer method (HVJ-liposome method). Previously, we used two different approaches for transfection into rat adult liver: direct injection into the liver and injection into the portal vein. Therefore, we used both approaches in the present study. First, we attempted direct injection into the liver (Table). Transfection of antisense ODNs with HVJ-liposome solution resulted in the inhibition of hepatic angiotensinogen production (P<.01), whereas there were no significant differences between sense and control groups. Next, we examined the transfection of ODNs with HVJ-liposome complex via the portal vein to deliver antisense ODNs into the whole liver. Fig 3a shows a typical example of Northern blot analyses of hepatic angiotensinogen mRNA treated with both antisense and sense ODNs. A reduction of hepatic angiotensinogen mRNA was observed on day 2 after transfection with antisense ODNs. Hepatic angiotensinogen mRNA level did not differ significantly between sense treatment and untreated groups (data not shown). The ratio of angiotensinogen mRNA to rat GAPDH mRNA was significantly decreased by the antisense treatment compared with the sense treatment group, as shown in Fig 3b (sense, 0.98±0.06 versus antisense, 0.55±0.06, P<.01). There was no evidence that transfection itself changed production of hepatic angiotensinogen, which is an acute phase protein, because there was no significant change in hepatic angiotensinogen content between untransfected and sense ODN–transfected rats (untransfected, 73.4±4.5 ng Ang I/g tissue versus ODN-transfected, 68.4±11.2 ng Ang I/g tissue, P=NS).
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We also measured plasma levels of angiotensinogen on days 0, 3, 5, and 7 after the injection of HVJ-liposome complex. Transfection of antisense ODNs resulted in a significant decrease in plasma angiotensinogen levels on days 3, 5, and 7 after injection compared with sense ODN treatment (P<.01, Fig 4a), consistent with Northern blot analysis. On day 3, the maximal decrease in angiotensinogen level was observed. We also assessed the effect of antisense treatment on mean BP. Mean BP began to decrease at 1 day after injection, and then it decreased from 172 mm Hg on day 0 to 154 mm Hg on day 2 after the injection of HVJ-liposome solution (P<.01). Transfection of antisense ODNs resulted in a significant decrease in BP level from day 1 to day 4 after injection compared with sense ODN treatment (P<.01, Fig 4b). To test whether the reduction of angiotensinogen affects plasma Ang II concentration, we measured plasma Ang II concentration after transfection of ODNs. Our results documented that the decrease in plasma Ang II concentration was observed on day 2 after antisense treatment (antisense group, 1.5±0.3 pg/mL; sense group, 2.3±0.1 pg/mL; P<.05, n=3). In contrast, sense and antisense treatment groups did not differ significantly on day 5 after transfection (antisense group, 2.2±0.2 pg/mL; sense group, 2.6±0.4 pg/mL; n=3). BP, plasma angiotensinogen level, and plasma Ang II concentration did not differ significantly between sense treatment and untreated groups (data not shown). Moreover, the results were not affected by hepatic dysfunction, as shown by the fact that there was no liver toxicity. Results of liver function tests showed no significant changes between ODN-transfected rats and untreated rats at 5 days after transfection (plasma aspartate aminotransferase: untransfected, 52±3.7 IU/L versus ODN-transfected, 54±4.5 IU/L; plasma alanine aminotransferase: untransfected, 26.0±5.2 IU/L versus ODN-transfected, 25.0±6.4 IU/L; total protein: untransfected, 6.2±0.5 IU/L versus ODN-transfected, 6.0±0.7 IU/L; total cholesterol: untransfected, 44.0±3.4 mg/dL versus ODN-transfected, 42.0±4.2 mg/dL; n=7-8). These results are consistent with our previous reports that significant histological change was not observed after injections of HVJ-liposome solution.17 18 20
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Discussion |
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Angiotensinogen is the precursor of the vasoactive peptide Ang II and is therefore speculated to be an important determinant of BP and electrolyte homeostasis.1 2 Recent genetic linkage studies, including work from our laboratory, suggest that angiotensinogen may be one of the candidate genes for hypertension.6 7 8 The observation that plasma angiotensinogen concentration correlates with BP also supports the possibility that circulating angiotensinogen plays an important role in the pathogenesis of hypertension.6 The role of angiotensinogen in BP regulation was previously demonstrated by the observation that angiotensinogen antibody administration resulted in the reduction of BP.30 Moreover, the acute administration of pure rat angiotensinogen to rats has been reported to increase BP.31 Since these previous studies did not change angiotensinogen production rates, it is important to note how angiotensinogen modulates BP. Here, we addressed two specific questions: (1) Does angiotensinogen have a role in BP regulation? and (2) is it possible to change BP level by manipulating circulating angiotensinogen level? For these aims, we used molecular biological techniques, a gene transfer method (HVJ-liposome method), and antisense technology. The HVJ-liposome method has been reported to be efficient for transfection into various organs such as the liver and vessel wall.17 18 19 20 Moreover, this method enhances the effectiveness and prolongs the half-life of antisense ODNs.21 22 Antisense technology is an innovative and attractive strategy for blocking the transcription or translation of specific genes.13 14 The combination of both molecular biological techniques may give us new information about the pathobiology of hypertension.
In this study, we initially evaluated three different sequences of ODNs against the region around the rat angiotensinogen transcription start site. As shown in Fig 2a, only antisense ODN 3 had an inhibitory effect on angiotensinogen production. No effects of other sets of antisense ODNs and scrambled ODNs supported the antisense-specific effects. Interestingly, antisense ODNs ("naked" ODNs) without HVJ-liposome complex had no inhibitory effects on angiotensinogen production (data not shown), indicating that the HVJ-liposome method is very effective for ODN delivery. Moreover, these effects were dose dependent without toxicity. Interestingly, we found that only antisense ODN 3 directed against the exon 1/intron 1 junction was effective in inhibiting angiotensinogen production. Thus, the effects of ODN 3 are most likely due to an inhibition of splicing of this first intron and retention of the transcript in the nucleus. This fact also implies that the site of activity of ODN 3 is in the nucleus, where splicing occurs. Previous studies have reported that ODNs that enter into the cytoplasm can easily translocate into the nucleus without any helper such as HMG-1. We also confirmed those previous observations using fluorescein isothiocyanate–labeled ODNs.22
Using these antisense ODN sequences, we examined two different approaches: direct multiple injections into the liver and injection into the portal vein for ODN delivery into the liver in vivo. Previously we reported the transfection of the human renin gene into the rat liver by direct multiple injections.20 In that report, overexpression of human renin gene in the whole liver was not necessary to achieve the biological effects of the transgene on BP regulation because the produced renin was circulating in plasma. However, for the aim of the present study, the delivery of antisense ODNs into the whole liver was ideal. Therefore, we used delivery into the liver with HVJ-liposome solution via the portal vein, although transfection of antisense ODNs by multiple injections into the liver also resulted in inhibitory effects on angiotensinogen production (Table). On day 2, we performed Northern blot analysis of angiotensinogen in the liver (Fig 3). These data revealed that mRNA production was decreased because of the specific effect of antisense ODNs injected into the portal vein. We also measured BP from 4 days before to 6 days after ODN injection. Mean BP decreased from 1 day after the injection of antisense ODNs (Fig 4b), consistent with the reduction of mRNA production of angiotensinogen in the liver. A decrease in plasma angiotensinogen level was also observed (Fig 4a). Mean BP decreased most markedly on day 2 and then increased to recover to the normal level on day 5. However, the effect of antisense treatment on the plasma angiotensinogen level lasted at least until day 7. Interestingly, a discrepancy between mean BP and plasma angiotensinogen level was observed. This was probably due to the existence of compensating systems such as the central nervous system. The data of plasma Ang II concentration also support this idea. As shown in this study, Ang II concentrations decreased on day 2 but recovered on day 5 when angiotensinogen level was still decreased.
A classic pharmacological approach to define the role of the RAS is to use specific inhibitors of RAS components, such as angiotensin-converting enzyme inhibitors and Ang II receptor antagonists. However, such an approach has disadvantages: (1) the difficulty in discriminating between systemic and local effects of the inhibitors, and (2) the lack of inhibitors or antagonists directed against angiotensinogen.32 We attempted to develop molecular genetic techniques for the study of the RAS in the pathogenesis of hypertension. As mentioned above, there is no specific inhibitor against angiotensinogen, whose only known physiological function is to serve as the precursor of Ang II and as the first step in angiotensin production. In addition, enzyme kinetic data suggest that the renin reaction is proportional to plasma angiotensinogen concentration. Alternatively, transgenic/gene-targeting technology is also very useful for study of the specific functions of targeted genes. However, that technology also has several disadvantages, such as its inability to exclude the developmental and systemic effects of transgenes and the need for a relatively long experimental period.30 Taken together, in vivo gene transfer techniques and/or antisense technology may take a place in the study of the pathogenesis of hypertension, as shown in the present study.
This study first demonstrated the utility of gene transfer and antisense technology for hypertension research, especially for evaluating the specific functions of RAS components. Overall, our results revealed that the reduction of circulating angiotensinogen level by antisense technology resulted in a decrease of high BP in SHR. As the effect of antisense ODNs on tissue angiotensinogen levels was not fully understood in this study, further studies are necessary to clarify the general role of angiotensinogen. Our preliminary data also showed that in vivo transfection of angiotensinogen antisense ODNs resulted in a transient decrease in BP, even in normotensive rats (15.6±4.7 mm Hg), consistent with angiotensinogen knockout mice.33 34 Taken together, these results suggest that angiotensinogen level is an important determinant of hypertension in SHR and point toward the potential of angiotensinogen as an antihypertensive target.
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Acknowledgments |
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This work was partially supported by grants from the Japan Research Foundation for Clinical Pharmacology and Kanae Foundation of Research for New Medicine. Ryuichi Morishita is the recipient of a Japan Vascular Disease Research Foundation Award. We thank M. Mashimoto and K. Zaitsu for their technical assistance.
Received January 25, 1995; first decision February 16, 1995; accepted April 7, 1995.
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 C. P. R. Klett, D. Anderson, M. Sholook, and J. P. Granger Antisense oligodeoxynucleotides directed against a novel angiotensinogen mRNA-stabilizing protein reduce blood pressure in spontaneously hypertensive rats Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2004; 287(3): R619 - R626. [Abstract] [Full Text] [PDF]
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C. P. R. Klett and J. P. Granger Physiological elevation in plasma angiotensinogen increases blood pressure Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2001; 281(5): R1437 - R1441. [Abstract] [Full Text] [PDF]
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M. I. Phillips Gene Therapy for Hypertension: The Preclinical Data Hypertension, September 1, 2001; 38(3): 543 - 548. [Abstract] [Full Text] [PDF]
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K. Ishikawa, S. Baba, T. Katsuya, N. Iwai, T. Asai, M. Fukuda, S. Takiuchi, Y. Fu, T. Mannami, J. Ogata, et al. T+31C Polymorphism of Angiotensinogen Gene and Essential Hypertension Hypertension, February 1, 2001; 37(2): 281 - 285. [Abstract] [Full Text] [PDF]
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S. I. Garcia, A. L. Alvarez, P. I. Porto, V. M. Garfunkel, S. Finkielman, and C. J. Pirola Antisense Inhibition of Thyrotropin-Releasing Hormone Reduces Arterial Blood Pressure in Spontaneously Hypertensive Rats Hypertension, February 1, 2001; 37(2): 365 - 370. [Abstract] [Full Text] [PDF]
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 C. H. Gelband, M. J. Katovich, and M. K. Raizada Current Perspectives on the Use of Gene Therapy for Hypertension Circ. Res., December 8, 2000; 87(12): 1118 - 1122. [Abstract] [Full Text] [PDF]
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 M. Gardon, M. K Raizada, M. J Katovich, K. H Berecek, and C. H Gelband Gene therapy for hypertension and restenosis Journal of Renin-Angiotensin-Aldosterone System, September 1, 2000; 1(3): 211 - 216. [PDF]
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 N. TOMITA, R. MORISHITA, H. Y. LAN, K. YAMAMOTO, M. HASHIZUME, M. NOTAKE, K. TOYOSAWA, B. FUJITANI, W. MU, D. J. NIKOLIC-PATERSON, et al. In Vivo Administration of a Nuclear Transcription Factor-{kappa}B Decoy Suppresses Experimental Crescentic Glomerulonephritis J. Am. Soc. Nephrol., July 1, 2000; 11(7): 1244 - 1252. [Abstract] [Full Text]
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 T. Tomita, H. Takano, N. Tomita, R. Morishita, M. Kaneko, K. Shi, K. Takahi, T. Nakase, Y. Kaneda, H. Yoshikawa, et al. Transcription factor decoy for NF{kappa}B inhibits cytokine and adhesion molecule expressions in synovial cells derived from rheumatoid arthritis Rheumatology, July 1, 2000; 39(7): 749 - 757. [Abstract] [Full Text] [PDF]
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 I.-J. Moon, K. Choi, Y.-K. Choi, J.-E. Kim, Y. Lee, A. D. Schreiber, and J.-G. Park Potent Growth Inhibition of Leukemic Cells by Novel Ribbon-type Antisense Oligonucleotides to c-myb1 J. Biol. Chem., February 18, 2000; 275(7): 4647 - 4653. [Abstract] [Full Text] [PDF]
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 L. H. Wang, X. Y. Yang, R. A. Kirken, J. H. Resau, and W. L. Farrar Targeted disruption of Stat6 DNA binding activity by an oligonucleotide decoy blocks IL-4-driven TH2 cell response Blood, February 15, 2000; 95(4): 1249 - 1257. [Abstract] [Full Text] [PDF]
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 X. Tang, D. Mohuczy, Y. C. Zhang, B. Kimura, S. M. Galli, and M. I. Phillips Intravenous angiotensinogen antisense in AAV-based vector decreases hypertension Am J Physiol Heart Circ Physiol, December 1, 1999; 277(6): H2392 - H2399. [Abstract] [Full Text] [PDF]
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 N. Makino, M. Sugano, S. Ohtsuka, S. Sawada, and T. Hata Chronic antisense therapy for angiotensinogen on cardiac hypertrophy in spontaneously hypertensive rats Cardiovasc Res, December 1, 1999; 44(3): 543 - 548. [Abstract] [Full Text] [PDF]
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T. Nishii, A. Moriguchi, R. Morishita, K. Yamada, S. Nakamura, N. Tomita, Y. Kaneda, A. Fukamizu, H. Mikami, J. Higaki, et al. Angiotensinogen Gene–Activating Elements Regulate Blood Pressure in the Brain Circ. Res., August 6, 1999; 85(3): 257 - 263. [Abstract] [Full Text] [PDF]
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M. K. Raizada, M. J. Katovich, H. Wang, K. H. Berecek, and C. H. Gelband Is antisense gene therapy a step in the right direction in the control of hypertension? Am J Physiol Heart Circ Physiol, August 1, 1999; 277(2): H423 - H432. [Full Text] [PDF]
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S. Tomita, N. Tomita, T. Yamada, L. Zhang, Y. Kaneda, R. Morishita, T. Ogihara, V. J. Dzau, and M. Horiuchi Transcription Factor Decoy to Study the Molecular Mechanism of Negative Regulation of Renin Gene Expression in the Liver In Vivo Circ. Res., May 14, 1999; 84(9): 1059 - 1066. [Abstract] [Full Text] [PDF]
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M. I. Phillips Is Gene Therapy for Hypertension Possible? Hypertension, January 1, 1999; 33(1): 8 - 13. [Full Text] [PDF]
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D. Mohuczy, C. H. Gelband, and M. I. Phillips Antisense Inhibition of AT1 Receptor in Vascular Smooth Muscle Cells Using Adeno-Associated Virus-Based Vector Hypertension, January 1, 1999; 33(1): 354 - 359. [Abstract] [Full Text] [PDF]
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R. Morishita, J. Higaki, N. Tomita, and T. Ogihara Application of Transcription Factor "Decoy" Strategy as Means of Gene Therapy and Study of Gene Expression in Cardiovascular Disease Circ. Res., June 1, 1998; 82(10): 1023 - 1028. [Abstract] [Full Text] [PDF]
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N. Makino, M. Sugano, S. Ohtsuka, and S. Sawada Intravenous Injection With Antisense Oligodeoxynucleotides Against Angiotensinogen Decreases Blood Pressure in Spontaneously Hypertensive Rats Hypertension, May 1, 1998; 31(5): 1166 - 1170. [Abstract] [Full Text] [PDF]
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 L.-J. Zhu, M. K. Bagchi, and I. C. Bagchi Attenuation of Calcitonin Gene Expression in Pregnant Rat Uterus Leads to a Block in Embryonic Implantation Endocrinology, January 1, 1998; 139(1): 330 - 339. [Abstract] [Full Text] [PDF]
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 P. Corvol and X. Jeunemaitre Molecular Genetics of Human Hypertension: Role of Angiotensinogen Endocr. Rev., October 1, 1997; 18(5): 662 - 677. [Abstract] [Full Text]
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D. Lu, M. K. Raizada, S. Iyer, P. Reaves, H. Yang, and M. J. Katovich Losartan Versus Gene Therapy : Chronic Control of High Blood Pressure in Spontaneously Hypertensive Rats Hypertension, September 1, 1997; 30(3): 363 - 370. [Abstract] [Full Text]
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M. I. Phillips, D. Mohuczy-Dominiak, M. Coffey, S. M. Galli, B. Kimura, P. Wu, and T. Zelles Prolonged Reduction of High Blood Pressure With an In Vivo, Nonpathogenic, Adeno-Associated Viral Vector Delivery of AT1-R mRNA Antisense Hypertension, January 1, 1997; 29(1): 374 - 378. [Abstract] [Full Text] [PDF]
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V. J. Dzau and M. Horiuchi In Vivo Gene Transfer and Gene Modulation in Hypertension Research Hypertension, December 1, 1996; 28(6): 1132 - 1137. [Abstract] [Full Text]
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M. Sugano and N. Makino Changes in Plasma Lipoprotein Cholesterol Levels by Antisense Oligodeoxynucleotides against Cholesteryl Ester Transfer Protein in Cholesterol-fed Rabbits J. Biol. Chem., August 9, 1996; 271(32): 19080 - 19083. [Abstract] [Full Text] [PDF]
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D. Wielbo, A. Simon, M. I. Phillips, and S. Toffolo Inhibition of Hypertension by Peripheral Administration of Antisense Oligodeoxynucleotides Hypertension, July 1, 1996; 28(1): 147 - 151. [Abstract] [Full Text]
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K. Tamura, S. Umemura, N. Nyui, T. Yamakawa, S. Yamaguchi, T. Ishigami, S.-i. Tanaka, K. Tanimoto, N. Takagi, H. Sekihara, et al. Tissue-Specific Regulation of Angiotensinogen Gene Expression in Spontaneously Hypertensive Rats Hypertension, June 1, 1996; 27(6): 1216 - 1223. [Abstract] [Full Text]
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R. Morishita, J. Higaki, N. Tomita, M. Aoki, A. Moriguchi, K. Tamura, K. Murakami, Y. Kaneda, and T. Ogihara Role of Transcriptional cis-Elements, Angiotensinogen Gene–Activating Elements, of Angiotensinogen Gene in Blood Pressure Regulation Hypertension, March 1, 1996; 27(3): 502 - 507. [Abstract] [Full Text]
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M. Schinke, M. Bohm, G. Bricca, D. Ganten, and M. Bader Permanent Inhibition of Angiotensinogen Synthesis by Antisense RNA Expression Hypertension, March 1, 1996; 27(3): 508 - 513. [Abstract] [Full Text]
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 F. K. Askari and W. M. McDonnell Antisense-Oligonucleotide Therapy N. Engl. J. Med., February 1, 1996; 334(5): 316 - 318. [Full Text] [PDF]
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