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(Hypertension. 1995;25:314-319.)
© 1995 American Heart Association, Inc.
Articles |
Antisense Inhibition of Hypertension in the Spontaneously Hypertensive Rat
From the University of Florida, College of Medicine, Department of Physiology.
Correspondence to M.I. Phillips, University of Florida, College of Medicine, Department of Physiology, JHMHC Box 100274, Gainesville, FL 32610-0274.
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Abstract |
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Abstract Phosphorothioated antisense oligodeoxynucleotide (ASODN) targeted to angiotensinogen mRNA was administered intracerebroventricularly in spontaneously hypertensive rats to test whether angiotensinogen reduction would lower their hypertensive blood pressures. The ASODN lowers hypertensive blood pressures to normotensive levels in spontaneously hypertensive rats; sense oligodeoxynucleotide had no effect. Administration of phosphorothioated ASODN produced a prolonged duration of lowered blood pressure. Injections of ASODN at the same dose that decreased hypertension when administered centrally did not result in blood pressure decreases when administered intra-arterially. Furthermore, angiotensinogen production was decreased in the brain stem and significantly decreased in the hypothalamus of the ASODN-treated rats (P<.05), supporting the concept of centrally mediated regulation of hypertension by an overactive brain angiotensin system. To determine the distribution of centrally administered oligodeoxynucleotides, fluorescein isothiocyanate–conjugated oligodeoxynucleotides were injected directly into the lateral ventricles. One hour later, oligodeoxynucleotides were distributed throughout the lateral and third ventricles, with tissue and cellular uptake observed in discrete cells at the injection site. This indicates that the oligodeoxynucleotides are taken up rapidly by brain cells and that they permeate the areas surrounding brain nuclei involved in central blood pressure regulation and volume homeostasis. The results confirm and extend our previous study with phosphodiester ASODN and show that phosphorothioation modification increases the duration of the response and is taken up in vivo. We conclude that with modification, ASODN inhibition of angiotensinogen mRNA translation can be used for a prolonged, profound decrease in mean arterial pressure in the spontaneously hypertensive rat through a central mechanism.
Key Words: hypertension, essential • angiotensin II • angiotensinogen • RNA, messenger • DNA, antisense
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Introduction |
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An accumulation of evidence supports the existence of local renin-angiotensin systems (RASs) that function separately from the classic peripheral RAS. Generation of angiotensins by the RAS is dependent on the glycoprotein precursor angiotensinogen. The brain has all the requisite components for a RAS, with particularly high concentrations of angiotensinogen. This system has been shown to be involved in the central regulation of blood pressure and volume homeostasis via areas in the hypothalamus and brain stem.1 2 3 4 In addition, an overactive brain RAS has been implicated in the development and maintenance of blood pressure in the spontaneously hypertensive rat (SHR), the animal model of essential hypertension.5 6 The generation of angiotensin II (Ang II) and the expression of Ang II receptors and angiotensinogen are higher in SHRs than in genetically related normotensive rats.7 8 9 Decreasing Ang II activity in brain with saralasin10 and more recently with the Ang II AT1 receptor blocker losartan7 11 lowered blood pressure in different SHR strains. Recent evidence in humans implicating a genetic variant of angiotensinogen in the development of essential hypertension and related forms of hypertension12 13 14 suggests that angiotensinogen may, in some circumstances, be a significant factor in hypertension.
Antisense oligodeoxynucleotides (ASODNs) have been used successfully to inhibit protein synthesis in a number of biological systems.15 16 17 This paradigm of gene regulation has many potential therapeutic applications. Advances in the understanding of the function, metabolism, and structure of these molecules have led to the development of antisense molecules with enhanced nuclease resistance and increased specificity, selectivity, and potency.17 Antisense regulation or attenuation of protein synthesis can be applied to any candidate gene with a known molecular sequence. Antisense molecules are short strands of DNA or RNA, usually 12 to 18 bases in length, that are synthesized to complement a target region of a candidate gene. The antisense molecule binds to its complementary region and, through a number of mechanisms, inhibits or attenuates gene expression.18 The success of ASODNs in the inhibition of protein synthesis in a number of biological systems presents a new approach to gene therapy and protein regulation. We hypothesized that central injections of ASODN targeted to angiotensinogen mRNA would decrease blood pressure in the SHR by modulating the centrally mediated regulation of blood pressure. We previously showed that central administration of phosphodiester ASODNs that target angiotensinogen mRNA decreased blood pressure in the SHR.19 Although blood pressure decreases in the SHR were profound enough to reduce blood pressure to normotensive levels, the duration was short. This could be attributed to the short half-life of phosphodiester oligodeoxynucleotides in biological fluids caused by extensive, rapid endonuclease and exonuclease degradation.20 In this study, we hypothesize that the administration of modified, phosphorothioated ASODNs to angiotensinogen will result in a more profound and prolonged decrease in blood pressure compared with the previously tested phosphodiester oligodeoxynucleotides. This modification enhances nuclease resistance.17 In addition, we hypothesize that the physiological response observed by central administration of ASODN is through modification of angiotensinogen levels in the brain.
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Methods |
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Materials and Methods
Animals
Adult male SHR (250 to 275g) were acquired from Harlan. The animals were kept in cages in a room with a 12-hour light-dark cycle. Animals were fed standard laboratory rat chow and tap water ad libitum. Each animal was anesthetized with sodium pentobarbital (65 mg/kg IP) and stereotaxically fitted with an intracerebroventricular stainless steel guide cannula (23 gauge). The stereotaxic coordinates were 1.0 mm lateral, 1.0 mm caudal to the bregma, and 5.0 mm below the skull surface. The animals were allowed to recover for 5 days before further surgical procedures or blood pressure measurements. Blood pressure was monitored by a direct pressure transducer through an in-dwelling catheter placed in the left carotid artery. Catheters were tunneled under the skin and exposed between the scapulae to prevent damage. Catheter patency was maintained with heparinized saline (100 U/mL). Animals were allowed to recover for 24 hours after catheterization before experimentation. All experimental protocols were approved by the Animal Care Committee of the University of Florida, Gainesville.
Oligodeoxynucleotides
ASODNs and sense oligodeoxynucleotides (SODNs) were synthesized
as 18-mers targeted to bases -5 to +13 of angiotensinogen mRNA
encompassing the AUG translation initiation codon. The strategy was to
interfere with ribosome assembly at the AUG codon and inhibit the
translation of the target protein. These oligodeoxynucleotides were
modified by backbone phosphorothioation. Oligodeoxynucleotides were
synthesized in the DNA Synthesis Laboratory, University of Florida. The
AS sequence to angiotensinogen mRNA was 5'-CCGTGGGAGTCATCACGG-3', and
the corresponding control S sequence was 5'-CCGTGATGACTCCCACGG-3'.
Fluorescently conjugated oligodeoxynucleotides were composed of the
same phosphorothioated sequences with conjugated fluorescein
isothiocyanate at both the 5' and 3' ends.
Radioimmunoassay for the Analysis of Angiotensinogen
Angiotensinogen secretion was measured by radioimmunoassay with
anti-angiotensinogen antiserum (R817) raised in rabbit against pure rat
angiotensinogen.21 Cross-reactivity was determined against
angiotensinogens from sheep, rabbit, donkey mouse, and humans with rat
albumin, Ang II, and tetradecapeptide. Angiotensinogen sample content
was measured from a standard curve of angiotensinogen diluted in medium
corresponding to the sample. The assay sensitivity was 0.3 ng per tube,
and the interassay and intra-assay variabilities were 14% and 9%,
respectively.
Determination of the Effects of Phosphorothioated ASODN to
Angiotensinogen mRNA on Mean Arterial Pressure
Groups of SHRs (n=6 to 8 per group) were administered a single
50-µg (10 µg/µL) dose of ASODN or SODN directly into the lateral
ventricle through the guide cannula. Baseline mean arterial pressure
(MAP) was established before injection through the implanted carotid
catheter. Blood pressure was monitored by a direct pressure transducer
and recorded on a Gould 2400 physiograph or a Digimed blood pressure
analyzer (Micro-med, Inc). MAP was monitored at 8 and 24 hours after
injection.
Determination of the Effects of Peripheral Administration of ASODN
on MAP
To determine whether the observed physiological responses were
due to central or peripheral effects of antisense, peripheral
injections of ASODN and SODN were administered intra-arterially. Groups
(n=3 to 6 per group) of male SHRs were cannulated
intracerebroventricularly, catheterized in the left carotid artery, and
allowed to recover before baseline MAP was established. Each group was
administered ASODN or SODN intra-arterially through the carotid
catheter. Oligodeoxynucleotide solution (50 µg) was administered in
200 µL isotonic saline solution. MAP was monitored continuously for
at least 8 hours.
Determination of the Effects of ASODN on Brain
Angiotensinogen
Three groups of SHRs (n=6 per group) were cannulated
intracerebroventricularly and administered a 50-µg dose of ASODN or
SODN or 5 µL isotonic saline. The animals were anesthetized and
transcardially perfused 24 hours later with 20 mL heparinized isotonic
saline. Brains were removed, and selected blocks were homogenized in 2
mL dH2O. Samples were centrifuged, and the supernatant was
lyophilized until radioimmunoassay procedures were carried out to
determine angiotensinogen levels.
Analysis of Fluorescein Isothiocyanate–Conjugated ASODN After
Central Administration
To determine the tissue distribution of the oligodeoxynucleotide
once it is injected into the lateral ventricles, male SHRs were
anesthetized with pentobarbital and injected intracerebroventricularly
with a microliter Hamilton syringe attached to the stereotaxic
apparatus. The coordinates used were the same as those for
intracerebroventricular cannulation. Rats were injected with either
ASODN or SODN (5 or 10 µg/µL) that was labeled at the 5' and 3'
ends with fluorescein. Injections were made slowly, and the injector
was left in position for 5 minutes after injection. Animals were left
for 1 hour and then perfused transcardially with saline, after which
the tissues were fixed with formaldehyde perfusion. Brains were then
removed and kept in 4% formaldehyde solution until cryostat
sectioning. Sections (50 µm) were obtained of the site of injection,
the lateral and third ventricle sites, including the hypothalamic and
circumventricular regions, and the brain stem. Sections were then
mounted and observed by laser scanning confocal microscopy to determine
oligodeoxynucleotide distribution and cellular uptake.
Statistical Analysis
All values were expressed as mean±SEM. Statistical analysis
was performed by ANOVA for treatment effect, and Duncan multiple-range
test was used for individual comparisons. Radioimmunoassay data and
intra-arterial data for individual time points were analyzed by
Student's independent t test. A probability value of <.05
was considered statistically significant.
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Results |
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The result of injecting ASODN to angiotensinogen mRNA was that blood pressures were reduced from hypertensive levels to normotensive levels in the ASODN-treated animals. Because of the variability in baseline MAP in the SHR model, all baseline pressures indicated hypertension as defined (above 150 mm Hg but ranged from 160 to 190 mm Hg). Therefore, blood pressure data were expressed as the change in MAP after oligodeoxynucleotide treatment. Fig 1 shows the effect of central injections of ASODN on MAP in the SHR. The MAP of the ASODN-treated animals was significantly decreased 8 hours after injection by -20.83±6.38 mm Hg; at 24 hours, this decrease was even greater, -37.14±7.69 mm Hg. There was no difference in the response to SODN after 8 and 24 hours (9.63±5.91 versus 6.67±5.91 mm Hg at 8 and 24 hours, respectively). ASODN administration significantly reduced MAP compared with SODN administration at the same time intervals (P<.01).
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Fig 2 shows the effect of intra-arterial administration of ASODN on MAP in a separate group of SHR. There was no significant difference in MAP between ASODN- and control-treated groups measured for more than 8 hours after injection.
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To determine the effects of centrally administered ASODN on angiotensinogen production in specific brain regions, another group of animals received intracerebroventricular injections of either 50 µg ASODN or SODN or 5 µL isotonic saline. Fig 3 shows the changes in angiotensinogen 24 hours after treatment. There was a decrease in angiotensinogen levels in the brain stems of ASODN-treated rats and a significant decrease in hypothalamic angiotensinogen levels after ASODN treatment (2474±195 ng/g) compared with SODN and saline control treatments (3486±261 and 3670±182 ng/g, P<.05). ASODN treatment did not result in changes in angiotensinogen levels in the cortex, midbrain, or cerebellum. Angiotensinogen levels were unaffected by control treatments of SODN or isotonic saline.
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The distribution of fluorescein isothiocyanate (FITC)–conjugated oligodeoxynucleotide was determined 1 hour after central injection by laser scanning confocal microscopy of brain sections. Fig 4 shows the region of the dorsal third and lateral ventricles (x2.5 objective). A strong fluorescent signal is observed around the injection site and within the ventricular system, particularly the dorsal third ventricle. A weaker fluorescent signal was also observed in the region of the third ventricle adjacent to the periventricular nuclei. Fig 5 shows the uptake of oligodeoxynucleotide into the surrounding tissue and into individual cells close to the injection site.
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Discussion |
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The results show that antisense inhibition of brain angiotensinogen mRNA reduced hypertension in the SHR. Chemical modification of the ASODN targeted to angiotensinogen results in a significant and prolonged decrease in MAP in SHRs after central administration of a single dose. The maximal decrease in MAP is greater than that observed with treatment of phosphodiester ASODN to angiotensinogen mRNA.19 In addition, the duration of action of the ASODN is extended by modification. MAP is still significantly decreased 24 hours after a single injection compared with the baseline MAP. By comparison, the duration of the physiological response elicited by a single injection of phosphodiester ASODN was only 3 hours.
The difference between phosphorothioated and phosphodiester ASODN can probably be attributed to the enhanced nuclease resistance of the phosphorothioated ASODN in conjunction with its subsequent activation of RNAse H.17 This combination would allow a larger fraction of the original dose to reach the ASODN site of action and prolonged stimulation of RNAse H owing to the nuclease resistance compared with the phosphodiester oligodeoxynucleotide. The prolonged half-life of the modified oligodeoxynucleotide also promotes a catalytic effect.17 20 As RNAse H degrades the mRNA strand of the ASODN-mRNA duplex, the ASODN strand is freed and able to bind to another strand of the target mRNA.
We hypothesized that central injections of ASODN targeted to angiotensinogen mRNA will decrease blood pressure in the SHR by reducing overactive brain angiotensin. Because of the existence of the blood-brain barrier, it would be expected that the centrally administered, negatively charged ASODN molecules would be unable to exert effects in the periphery. To determine whether the observed physiological responses were due to central effects of antisense, peripheral injections of ASODN and SODN were administered at the same dose that produced effects when administered centrally. This dose did not affect MAP in the SHR. If any or all the central ASODN were working peripherally, no more than the total dose of 50 µg could leak into the blood, and because we showed that this dose is ineffective intra-arterially, we conclude that the antihypertensive effect is due to central inhibition of brain angiotensinogen by the ASODN.
To determine whether translational inhibition of angiotensinogen synthesis was occurring, brain angiotensinogen levels were measured by radioimmunoassay. Angiotensinogen was shown to be decreased in the brain stem and significantly decreased in the hypothalamus of ASODN-treated animals compared with SODN- and saline-treated controls. No changes were observed in peripheral angiotensinogen levels. Thus, the antisense inhibited the synthesis of the target protein angiotensinogen in specific brain regions involved in the central regulation of blood pressure. Total inhibition of angiotensinogen was not observed, although the fall in blood pressure was profound. This may be accounted for by the method of analysis because blocks of tissue were assayed and may include nuclei or cells that were not reached or affected by the ASODN. Furthermore, angiotensinogen is secreted constitutively and not stored intracellularly in secretory granules. Hence, the effect of secretion may be more profound than indicated by the fall in tissue angiotensinogen content. In any event, the hypothesis of an overactive brain RAS would not require that angiotensinogen synthesis be abolished but only that it be reduced sufficiently to induce a fall in blood pressure to normotensive levels.13 This is supported by the lack of response of ASODN on angiotensinogen mRNA in Wistar-Kyoto (WKY) WKY rats.19 Because WKY rats did not respond to phosphodiester oligodeoxynucleotides, this study deals only with the response in SHRs.
By using a combination of FITC-conjugated ASODN and confocal microscopy, we have shown the distribution of oligodeoxynucleotides after central administration. The tissue uptake was greatest around the site of injection. The ventricular distribution and the angiotensinogen data suggest that the ASODN is able to reach the hypothalamic brain sites and the brain stem sites involved in central regulation of blood pressure. The distribution of centrally administered FITC-conjugated SODN was no different from the distribution of the FITC-conjugated ASODN. Further work will show whether specific sites of injection are more effective.
This study shows several new findings. First, the phosphorothioation of the ASODN to angiotensinogen resulted in a significant decrease in MAP comparable to that of the unmodified phosphodiester ASODN. Second, significant blood pressure decreases were achieved with a single 50-µg dose compared with three doses of the unmodified oligodeoxynucleotide required to produce the same effect. In addition, the duration of the response was significantly prolonged compared with that of the unmodified ASODN. Third, the study shows the effect of ASODN on angiotensinogen production in the hypothalamus, a brain area thought to be involved in central cardiovascular regulation and fluid homeostasis. Fourth, peripheral administration of ASODN resulted in no decrease in blood pressure at a dose that has physiological effects when administered centrally. This suggests that the observed effects are mediated through central and not peripheral mechanisms. The observed decrease in angiotensinogen indicates successful translational inhibition of the candidate gene product by the modified ASODN constructed to complement angiotensinogen mRNA. From these data, suitably modified ASODN would appear to have the potential to produce long-term reduction of hypertension by specifically inhibiting synthesis of a candidate gene product. The advantages of this approach for future development are the specificity, decreased toxicity, effectiveness, and potential for prolonged benefit with fewer doses.
All the genes of the RAS have been shown to be expressed in the brain. Angiotensinogen mRNA has been shown in the highest concentrations in the brain stem and midbrain but also in the cerebellum, diencephalon, and cortex.3 Renin mRNA has been shown to be present in rat brain, although in relatively low abundance.2 23 Angiotensin-converting enzyme mRNA has been localized to the choroid plexus, caudate putamen hippocampus, and cerebellum.24 Ang II has been measured by radioimmunoassay and high-performance liquid chromatography in the brain. The highest density was found in the paraventricular nucleus and supraoptic nuclei of the hypothalamus, but Ang II is also distributed widely in fibers and neurons in the hypothalamus, hippocampus, nucleus tractus solitarius, and cerebellum.25 The SHR has a hyperactive brain RAS compared with normotensive WKY rats, as demonstrated by higher Ang II levels,25 renin activity,26 angiotensin receptors,1 and angiotensinogen mRNA.7 Previous studies showed that decreasing brain Ang II activity with antagonists or converting-enzyme inhibitors decreased the blood pressure in SHRs but not in WKY rats.10 27 28 The present study has shown that the inhibition of brain angiotensinogen is also effective in decreasing the hypertension of SHR without affecting the blood pressure of WKY rats. There are some discrepancies between laboratories using central losartan, the AT1 receptor inhibitor in SHR. Pare et al11 and Yang et al29 found reductions in blood pressure and SHR with losartan, but DePasquale et al30 did not. Kawano et al31 showed that a low dose of losartan was ineffective but that a high dose produced a significant decrease in blood pressure. Because there are various reasons for the differences in these studies, including dose, drug metabolism, and time of measurement, and because they tested only the AT1 receptor, the results should not be used to dismiss the hypothesis of a RAS being involved in SHR hypertension. It is not known whether brain Ang II is synthesized intracellularly, extracellularly, or both. We, and others, have proposed that Ang II is produced extracellularly because of the extracellular location of angiotensin-converting enzyme.4 Studies in neurons and glial cells indicate that angiotensin is taken up from these paracrine sites of synthesis, stored, and released from neurons.25 ASODN to angiotensinogen mRNA would be effective in reducing Ang II, and our results show that ASODN significantly inhibits the synthesis of brain angiotensinogen. Therefore, the observed effects can be reasonably explained by a decrease in Ang II generated within the brain.
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Acknowledgments |
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Dr Sernia was on sabbatical from the Department of Physiology and Pharmacology, University of Queensland, Australia. We wish to thank Dan Tran, Shawn Toffolo, and David Kerr for their expert technical assistance. This study was supported by American Heart Association, Florida Affiliate Fellowships (Drs Wielbo and Gyurko) and National Institutes of Health grant HL-27334 (Dr Phillips).
Received June 8, 1994; first decision August 31, 1994; accepted November 7, 1994.
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S. Kagiyama, A. Varela, M. I. Phillips, and S. M. Galli Antisense Inhibition of Brain Renin-Angiotensin System Decreased Blood Pressure in Chronic 2-Kidney, 1 Clip Hypertensive Rats Hypertension, February 1, 2001; 37(2): 371 - 375. [Abstract] [Full Text] [PDF]
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B. Kimura, D. Mohuczy, X. Tang, and M. I. Phillips Attenuation of Hypertension and Heart Hypertrophy by Adeno-Associated Virus Delivering Angiotensinogen Antisense Hypertension, February 1, 2001; 37(2): 376 - 380. [Abstract] [Full Text] [PDF]
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J. Monti, M. Schinke, M. Bohm, D. Ganten, M. Bader, and G. Bricca Glial angiotensinogen regulates brain angiotensin II receptors in transgenic rats TGR(ASrAOGEN) Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2001; 280(1): R233 - R240. [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. 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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S. Nakamura, A. Moriguchi, R. Morishita, K. Yamada, T. Nishii, N. Tomita, M. Ohishi, Y. Kaneda, J. Higaki, and T. Ogihara Activation of the Brain Angiotensin System by In Vivo Human Angiotensin-Converting Enzyme Gene Transfer in Rats Hypertension, August 1, 1999; 34(2): 302 - 308. [Abstract] [Full Text] [PDF]
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 D. Y. Li, Y. C. Zhang, M. I. Philips, T. Sawamura, and J. L. Mehta Upregulation of Endothelial Receptor for Oxidized Low-Density Lipoprotein (LOX-1) in Cultured Human Coronary Artery Endothelial Cells by Angiotensin II Type 1 Receptor Activation Circ. Res., May 14, 1999; 84(9): 1043 - 1049. [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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B. S. Huang and F. H. H. Leenen Both Brain Angiotensin II and "Ouabain" Contribute to Sympathoexcitation and Hypertension in Dahl S Rats on High Salt Intake Hypertension, December 1, 1998; 32(6): 1028 - 1033. [Abstract] [Full Text] [PDF]
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B. C. Yang, M. I. Phillips, Y. C. Zhang, B. Kimura, L. P. Shen, P. Mehta, and J. L. Mehta Critical Role of AT1 Receptor Expression After Ischemia/Reperfusion in Isolated Rat Hearts : Beneficial Effect of Antisense Oligodeoxynucleotides Directed at AT1 Receptor mRNA Circ. Res., September 7, 1998; 83(5): 552 - 559. [Abstract] [Full Text] [PDF]
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 B. C. Yang, M. I. Phillips, D. Mohuczy, H. Meng, L. Shen, P. Mehta, and J. L. Mehta Increased Angiotensin II Type 1 Receptor Expression in Hypercholesterolemic Atherosclerosis in Rabbits Arterioscler. Thromb. Vasc. Biol., September 1, 1998; 18(9): 1433 - 1439. [Abstract] [Full Text] [PDF]
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J.-F. Peng, B. Kimura, M. J. Fregly, and M. I. Phillips Reduction of Cold-Induced Hypertension by Antisense Oligodeoxynucleotides to Angiotensinogen mRNA and AT1-Receptor mRNA in Brain and Blood Hypertension, June 1, 1998; 31(6): 1317 - 1323. [Abstract] [Full Text] [PDF]
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 B. Yang, D. Li, M I. Phillips, P. Mehta, and J. L Mehta Myocardial angiotensin II receptor expression and ischemia-reperfusion injury Vascular Medicine, May 1, 1998; 3(2): 121 - 130. [Abstract] [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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D. Lu, H. Yang, and M. K. Raizada Attenuation of ANG II actions by adenovirus delivery of AT1 receptor antisense in neurons and SMC Am J Physiol Heart Circ Physiol, February 1, 1998; 274(2): H719 - H727. [Abstract] [Full Text] [PDF]
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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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