(Hypertension. 1997;30:363.)
© 1997 American Heart Association, Inc.
Articles |
Losartan Versus Gene Therapy
Chronic Control of High Blood Pressure in Spontaneously Hypertensive Rats
From the Department of Physiology, College of Medicine (D.L., M.K.R., P.R., H.Y.), and the Department of Pharmacodynamics, College of Pharmacy (S.I., M.J.K.), University of Florida, Gainesville.
Correspondence to Mohan K. Raizada, PhD, Professor and Associate Dean for Graduate Education, Department of Physiology, College of Medicine, University of Florida, PO Box 100274, Gainesville, FL 32610.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract Interruption of the renin-angiotensin system by pharmacological manipulations attenuates high blood pressure (BP) in the spontaneously hypertensive rat (SHR). However, these agents, such as losartan, need to be administered daily to maintain effective BP control. Therefore, we have hypothesized that a genetic intervention in the expression of angiotensin type 1 receptor (AT1R) should attenuate development of hypertension on a long-term basis in SHR. A retroviral-mediated AT1R antisense cDNA gene delivery system (LNSV–AT1R-AS) was used to test this hypothesis and to compare its BP-lowering effects with those of losartan. Introduction of LNSV–AT1R-AS into 5-day-old Wistar-Kyoto rats and SHR resulted in a robust expression of AT1R antisense (AS) within 3 days and persisted for at least 30 days. This expression was associated with a selective attenuation of high BP in SHR by 25 to 30 mm Hg. Although basal lowering of BP was exclusive to SHR, the angiotensin II (Ang II) pressor response was significantly reduced in all LNSV–AT1R-AS–treated rats. The decreased response to Ang II was associated with a similar attenuation of Ang II–induced dipsogenic responses in both strains of rats. The BP-lowering effects of LNSV–AT1R-AS treatment and losartan treatment were similar and primarily observed in SHR. However, the antihypertensive effect lasted less than 24 hours in losartan-treated SHR compared with 90 days in LNSV–AT1R-AS–treated SHR. In addition, losartan was unable to further lower BP in LNSV–AT1R-AS–treated SHR. Collectively, these results suggest that both losartan and LNSV–AT1R-AS treatment produces an antihypertensive response selectively in SHR that is mediated by interruption of AT1R function. However, a single, acute genetic treatment with LNSV–AT1R-AS can result in long-term control of high BP at a similar level of effectiveness as losartan, without altering plasma Ang II levels.
Key Words: AT1 receptor • hypertension • gene transfer • gene therapy • blood pressure • angiotensin II
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Overwhelming evidence indicates that the RAS is important in the development and maintenance of hypertension in both essential hypertensive patients and animal models of hypertension, including SHR.1 2 3 When the RAS is interrupted by pharmacological manipulations, systemic BP and other cardiovascular complications of high BP are reduced.4 5 6 7 8 Recent evidence has suggested that there is a critical period for the development of hypertension in SHR. This evidence stems from studies which showed that early treatment of young SHR with various inhibitors of angiotensin-converting enzyme can prevent the expression of hypertension even after the treatment is discontinued.9 10 11
Numerous pharmacological agents are available for successful treatment of essential hypertension in humans. Many of these agents act by either blocking the formation of Ang II or, more recently, by antagonizing the AT1R. Among them is a newly developed AT1R antagonist, losartan, which has been proven as a highly reliable antihypertensive drug without significant side effects.12 13 14 15 Since it has been demonstrated that the AT1R-encoding gene polymorphism is highly coupled with hypertension16 and that losartan is a successful antihypertensive agent, it would appear logical that the AT1R is an important target for therapeutic intervention in Ang II–dependent forms of hypertension.13 14 These observations, coupled with our in vitro data, which showed that the delivery of AT1R-AS by a retrovirally mediated gene delivery system can selectively attenuate cellular actions of Ang II in the neurons of SHR,17 18 indicate that a gene targeting for the AT1R could be effective in the control of hypertension.
This view is further supported by recent observations that intracerebroventricular19 20 21 or peripheral22 23 administration of antisense oligonucleotides to the RAS decreases high BP in SHR. Thus, we hypothesized that in vivo genetic intervention in the expression of the RAS by retrovirally mediated gene delivery should attenuate the development of hypertension on a long-term basis and possibly even prevent the genesis of hypertension in SHR. The observations presented here show this to be the case and compare advantages of gene therapy with conventional losartan therapy.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Animals
WKY and SHR bred from our own animal colony were used for this study. The original source of the breeding animals was purchased from Harlan Sprague Dawley, Inc (Indianapolis, Ind). Five days after birth, the offspring were removed from their mothers and divided into three experimental groups: saline vehicle control; an LNSV virus control (virus administered without the antisense); and the virus treatment containing the AT1R antisense cDNA (LNSV–AT1R-AS). Treatment consisted of lightly anesthetizing the animals with methoxyflurane (Metofane, Pittman-Moore) and injecting, via a cardiac route, 10 µL of either physiological saline or a bolus of 1x109 plaque-forming units of viral particles (LNSV) with or without the incorporation of the AT1R-AS. After injection, animals were tagged (right ear clip for LNSV, left ear clip for LNSV–AT1R-AS, and no clip for the saline controls), lightly coated with peanut oil, and returned to their mothers until weaned. This treatment resulted in a very successful rate of acceptance of the pups by their mothers, and there was no observable difference in the survival rate (95% 24 to 48 hours after injection) among the different treatment groups. After 21 days of weaning, animals were group housed in an animal room maintained at 25±2°C with a 12:12 light:dark cycle. All animals were maintained on Richmond standard laboratory rodent diet and water ad libitum. There were no treatment effects on the growth rate or body weights of the animals. All animal procedures were preapproved by the Institutional Animal Care and Use Committee (IACUC), and all procedures followed were in accordance with the institutional guidelines.
Preparation of Viral Particles Containing the
AT1R-AS
Delivery of the AT1R-AS was performed by the use of
a retroviral vector, LNSV. This system has been successfully used for
delivering the AT1R-AS to brain cells in
culture.17 18 The AT1R-AS
(nucleotides -132 to +1128) was chosen as a result of
previous studies demonstrating that this AT1R-AS blocked
the functional aspects of both AT1A and AT1B
receptor subtypes.17 18 The AT1R-AS was cloned
in the LNSV that was kindly provided by Geoffrey Owens, University of
Colorado Health Science Center. LNSV is a retroviral vector of 6236 bp
derived from pLNL6 and contains long terminal repeats and
an internal simian virus 40 promoter that controls the expression of
the AT1R-AS. The vector was transfected into a packaging
cell line (PA317, American Type Culture Collection). After selection by
G418, the medium containing viral particles that expressed
AT1R-AS (LNSV–AT1R-AS) was collected and used
for the animal experiments. Viral particles that did not contain
AT1R-AS were used as the vector control (LNSV).
Measurement of AT1R-AS Transcripts
On days 3, 10, 30, and 60 after administration of
AT1R-AS, Ang II target tissues (heart, kidney, adrenals,
and mesenteric arteries) were harvested from control, LNSV-treated, and
LNSV–AT1R-AS–treated rats to determine the expression of
AT1R-AS transcript by a previously described
semiquantitative RT-PCR technique.17 18 24 Briefly, the
poly(A+) RNA from the tissues was isolated using Dynal
beads and subjected to a reverse transcription reaction with the use of
an AT1R sense primer (5'-CTTTCTTCTCAATCTCGCCTTGG-3'). This
procedure was followed by 18 cycles of PCR in the presence of 1 µCi
[32P]dCTP and both the AT1R sense primer and
antisense primer (5'-CCAGAAAGCCGTAGAACAGAGGG-3'). PCR products were
analyzed by polyacrylamide gel electrophoresis and
autoradiography.
Physiological Measurements
Indirect systolic BPs were measured at regular intervals
by the tail-cuff method25 in all animals from 33 to 78
days after treatment. At 40 and 80 days of age, the in vivo response to
Ang II was assessed by a dipsogenic response as previously
described.26 Briefly, rats were placed in individual
stainless steel metabolic cages fitted with 100-mL
graduated spillproof bottles (Bioserve) filled with tap water. Animals
were allowed to acclimate to the cages overnight. All dipsogenic
measurements were made between 9 AM and 1 PM in
a room maintained at 25±1°C. A 1-hour basal water intake was made by
subtracting the difference in water volume in the bottles before and
after the administration of the saline vehicle (1 mL/kg body weight)
subcutaneously. All animals were then administered Ang II (Research
Biochemicals International) at a concentration of 150 µg/kg,
subcutaneously, and water intake was measured for the subsequent hour.
Water intake was expressed as milliliters consumed per kilogram body
weight. No food was present during the dipsogenic study.
At the conclusion of the study, animals from each of the groups underwent carotid artery and jugular vein cannulations as previously described.27 Briefly, animals were anesthetized with a rodent cocktail containing ketamine (100 mg/mL) and xylazine (20 mg/mL), which was administered intramuscularly (0.7 mg/kg). A silicone elastomer catheter (Helix Medical) was implanted into the jugular vein for drug infusion, and the carotid artery was cannulated with a PE-50 catheter (Clay Adams) for direct BP determination. Both catheters were exteriorized between the shoulder blades, filled with a heparin solution (10 U/mL; Elkins-Sinn, Inc), and sealed with stylets. After a recovery period of 24 to 48 hours, direct BP was recorded in free-moving, nonrestrained animals with a pressure transducer coupled to a Digi-Med BP analyzer (Micro-Med). After a 30-minute equilibration period, the pressor response to Ang II (0.005 to 0.32 mg/kg, administered intravenously in a volume of 250 µL/kg body weight) was determined for each animal. EC50 values, concentrations producing 50% of the maximal response for Ang II, were calculated from the dose-response curves for the individual animals.
In an additional group of 3-month-old SHR and WKY, the effects of acute losartan were evaluated. All animals underwent surgical implantation of jugular and carotid cannulation as previously described.27 On the day of the direct mean BP measurement, in the unrestrained awake animal, after a 60-minute equilibration period, animals were challenged with Ang II (0.4 µg/kg) intravenously. BP was increased within 10 to 40 seconds, and after recovery to baseline, 0.9% physiological saline (Abbott Laboratories; 1 mL/kg) was administered intravenously as the control. Animals were then administered losartan (10 mg/kg) intravenously, and the mean BP and pressor response to Ang II were determined 1, 2, 3, 4, and 24 hours after administration of the losartan (DuPont-Merck Pharmaceutical Co).
Plasma Ang II levels were measured with the use of a radioimmunoassay kit (American Laboratory Products Co). Samples were obtained from trunk blood after decapitation, which occurred 3 to 4 hours after intravenous administration of saline (1 mL/kg) or losartan (10 mg/kg). Blood samples were centrifuged at 4°C, and plasma was separated and frozen until assayed. All samples were collected in EDTA-chilled tubes (Becton Dickinson) containing 1,10 phenanthroline (Sigma Chemical Company).
Ang II Receptor Autoradiography
Adrenals from rats 100 days after LNSV or
LNSV–AT1R-AS injections were frozen at -20°C and 20-mm
sections were prepared on gelatin-coated slides essentially as
described previously.28 Sections were preincubated for 30
minutes at room temperature with 50 mmol/L NaCl, 5
mmol/L EDTA, and 0.1 mmol/L bacitracin. This
procedure was followed by incubation with 100 pmol/L
125I-[Sar1,Ile8]-Ang II for 60
minutes at room temperature in the absence or presence of 1
µmol/L losartan or 1 µmol/L PD123319.
After washing and air drying, slides were exposed to X-ray film for 3
days and the autoradiograms obtained by developing the
film.
Measurement of AT1Rs
Left ventricles of hearts from rats treated with LNSV or
LNSV–AT1R-AS for 4 months were collected and the
homogenates prepared by electric
homogenizer (Tekmar Co). Membrane fractions were
prepared essentially as described elsewhere.29 Membranes
containing 60 µg protein were incubated with the binding buffer
(25 mmol/L Tris, 10 mmol/L MgCl2,
10 µg/mL bacitracin, 500 µmol/L DTT, and 0.2%
BSA, pH 7.5) in a final volume of 200 µL. The binding reaction was
started at room temperature by the addition of 0.3 nmol/L
125I-[Sar1,Ile8]-Ang II
containing 10 pmol/L to 100 nmol/L losartan for
60 minutes. Bound
125I-[Sar1,Ile8]-Ang II to
membranes was collected on Whatman GF/B filters. After washing with
ice-cold PBS, filters were dried and radioactivity was counted by a
Beckman Biogamma Counter (Beckman Instruments, Inc) essentially as
described previously.30 AT1R specific binding
was calculated by subtracting the radioactivity bound in the presence
of losartan from that bound in the absence of losartan.
Each data point was the mean of triplicates, and the experiment was
repeated three times. Scatchard analysis of these
competition-inhibition experiments was carried out as described
previously.31
Statistics
All results are expressed as mean±SE. Indirect BP measurements
were performed on 8 to 10 animals per group and analyzed by
repeated-measures ANOVA. A similar number of rats were used in all
other experiments unless stated otherwise. Direct mean BPs were
analyzed by ANOVA. For all statistical analysis,
differences between individual groups were compared using a Fisher’s
protected least significant difference posthoc test, and values of
P
.05 were considered statistically significant. PCR bands
corresponding to AT1R-AS were quantitated, normalized for
ß-actin mRNA, and presented as relative absorbance
essentially as previously described.24
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Effects of LNSV–AT1R-AS on Development of High BP in SHR
The effect of LNSV–AT1R-AS administration on indirect BP was measured as a function of development, and data are summarized in Fig 1. Two-way analysis revealed a significant effect of time and LNSV–AT1R-AS treatment on BP. No significant effect of LNSV–AT1R-AS treatment on BP of WKY was observed. The BP values of the LNSV–AT1R-AS–treated SHR were not significantly different from those observed in WKY treated with either saline or LNSV alone. Comparison of these data revealed no significant difference in BP between LNSV- and LNSV–AT1R-AS–treated WKY and SHR observed before 33 days of age. However, the average BPs over the 78 days in control and LNSV-treated SHR were significantly higher than their WKY counterparts (125.6±2.8 and 127.0±2.7 mm Hg for SHR versus 110.8±1.7 and 110.8±1.9 mm Hg for WKY, respectively). LNSV–AT1R-AS treatment reduced the elevated BP in SHR as early as 33 days, which was persistent throughout the measurement period. As a result, the mean BP over the 78-day period was significantly lower in LNSV–AT1R-AS–treated SHR (108.8±3.1 mm Hg) than in saline (125.6±2.8 mm Hg) or LNSV-treated (127.0±2.7 mm Hg) SHR controls (Fig 1) and not significantly different from any of the WKY groups. Direct mean BPs determined 90 days after administration of LNSV–AT1R-AS in unrestrained animals from all six groups are presented in Table 1. Mean BP was significantly elevated in control and LNSV-treated SHR compared with all other groups. No significant decrease in mean BP was observed between control and LNSV-treated SHR (P<.4). LNSV–AT1R-AS treatment caused a significant decrease in mean BP in SHR, which resulted in values similar to those obtained from the WKY groups. No such decrease was observed in LNSV–AT1R-AS–treated WKY compared with their control groups. These data indicated that LNSV–AT1R-AS treatment caused a selective antihypertensive effect in SHR, which persisted for at least 90 days.
|
), saline (
), or LNSV–AT1R-AS (•).
Indirect BP (mm Hg) was determined at 33, 39, 43, 49, 60, 67, and 78
days after treatment. LNSV- and saline-treated SHR developed
significantly higher BP than that observed in WKY. *Significantly lower
BP (P<.05) in the LNSV–AT1R-AS–treated SHR
compared with the saline- and LNSV-treated SHR.
|
AT1R-AS expression in various peripheral Ang II target tissues was determined by RT-PCR. Data in Fig 2 show that a robust expression of AT1R-AS was seen in heart and kidney as early as 3 days. The expression was maintained at detectable levels up to 30 days, and by 60 days no significant expression of AT1R-AS was observed (Fig 2). A similar pattern of expression of AT1R-AS was observed in other AT1R target tissues, such as adrenals and mesenteric artery (data not shown). These observations, coupled with BP data, suggest that although AT1R-AS expression is comparable in both strains, the antihypertensive response is observed only in SHR and not in WKY.
|
The Ang II–induced dipsogenic response was examined to evaluate a noninvasive physiological response to Ang II in LNSV–AT1R-AS–treated rats. Fig 3 presents data for 40 days (A) and 80 days (B) posttreatment. Administration of Ang II caused an average of 10 to 12 mL · kg-1 · h-1 increase in water intake in LNSV-treated, 40-day-old WKY and SHR. Control rats treated with saline consumed comparable amounts of water as the LNSV-treated rats. LNSV–AT1R-AS treatment attenuated Ang II–induced water intake by 70% to 75% in WKY and SHR (P<.02). There was no dipsogenic effect in LNSV–AT1R-AS–treated animals from both rat strains. A similar pattern of Ang II effects was observed after 80 days of treatment (Fig 3B). However, a significant (P<.05) strain effect was observed in these older SHR, in that basal water intake was elevated in SHR compared with WKY rats at 80 days. The Ang II–induced dipsogenic responses were comparable to those observed in 40-day-old rats. Collectively, these data indicate that AT1R-mediated responsiveness was significantly attenuated 40 days after administration of LNSV–AT1R-AS and that this attenuation is maintained throughout 80 days of treatment in both strains of rats.
|
Next, we compared the effect of the pressor response to Ang II in LNSV- and LNSV–AT1R-AS–treated WKY and SHR. Rats were challenged with several doses of intravenously administered Ang II while BP was recorded. Repeated-measures ANOVA revealed that Ang II increased BP in all groups in a dose-dependent manner (Table 2). Maximal increases in BP of 36.0±3.1 mm Hg and 44.6±2.8 mm Hg were observed in LNSV-treated WKY and SHR, respectively. The maximal pressor response to Ang II was significantly higher in SHR than in WKY. LNSV–AT1R-AS treatment resulted in a significant decrease in Ang II stimulation of BP in both strains of rats compared with their control counterparts. The reduced pressor responses in the LNSV–AT1R-AS–treated SHR were similar to or lower than those achieved in the WKY groups. The decrease in responsiveness was not due to changes in EC50 (Table 2), suggesting that the sensitivity of AT1R was not altered by LNSV–AT1R-AS treatment. However, since maximal response to the pressor effects of Ang II was reduced, it would suggest that LNSV–AT1R-AS treatment reduced the numbers of AT1R.
|
Comparison of Antihypertensive Effects of LNSV–AT1R-AS
With Losartan
The next series of experiments was carried out with
losartan to compare the antihypertensive effects of this
AT1R antagonist with the gene therapy approach.
Fig 4 shows that intravenous
administration of losartan in unrestrained SHR resulted in a
fall of mean BP. Within 2 hours of losartan administration, a
27.2±6.2 mm Hg lowering of BP in SHR to the level that was no
longer different from that observed in WKY control was observed. This
decrease in BP in SHR was significantly different from pretreatment BP.
The maximal lowering of BP with a single dose of losartan was
apparent by 2 hours and was maintained at this level up to 4 hours.
Within 24 hours, BP was no longer different from pretreatment levels in
SHR. In contrast, losartan had no significant effect in
lowering mean BP in WKY throughout the measurement period (Fig 4).
These data suggest that, similar to LNSV–AT1R-AS
treatment, losartan selectively lowers BP in SHR. However,
losartan’s antihypertensive response was short-lived compared
with LNSV–AT1R-AS treatment.
|
Pressor response to a single dose of Ang II was assessed in the same animals before and after administration of losartan. Before administration of losartan, Ang II significantly elevated mean BP in both WKY and SHR (Fig 5). A greater pressor response (26.8±3.1 mm Hg) was observed in SHR than in WKY (15.9±4.3 mm Hg). Within 1 hour after administration of losartan, the pressor responses to Ang II in both WKY and SHR were completely blocked. In SHR, the pressor response was significantly attenuated for 3 to 4 hours after a single injection of losartan and fully recovered 24 hours post-losartan. In contrast to the lack of effect on mean BP in WKY, the pressor response to Ang II was significantly attenuated within 1 hour of losartan treatment and maintained for 4 hours. As with SHR, the pressor response was normalized 24 hours after initial administration of losartan (Fig 5). These data suggest that the Ang II–induced pressor response was significantly lowered in both WKY and SHR in a transient manner.
|
Plasma Ang II levels were measured in losartan and LNSV–AT1R-AS–treated rats in view of previous indications of an increased Ang II level by losartan therapy. We found that within 4 hours post–losartan treatment, plasma Ang II levels were 34-fold and 65-fold higher than in saline-treated WKY and SHR. In contrast, LNSV–AT1R-AS treatment had no significant effect on plasma Ang II levels (4 to 5 pg/mL, n=4).
The effect of losartan on mean BP in control, LNSV-, and LNSV–AT1R-AS–treated SHR was measured to determine whether both losartan and LNSV–AT1R-AS target AT1R. Table 3 shows that losartan treatment significantly lowered BP in both control and LNSV-treated SHR. In contrast, losartan had little effect on lowering BP in LNSV–AT1R-AS–treated SHR or WKY. This treatment has been shown to lower BP in SHR by 25 to 30 mm Hg (Fig 4). Thus, the data demonstrate that both losartan and LNSV–AT1R-AS treatments lower BP by antagonizing AT1R activity, the former on a temporary and the latter on a long-term basis.
|
Finally, we studied the effect of LNSV–AT1R-AS treatment on the binding of 125I-[Sar1,Ile8]-Ang II. Fig 6A shows a competition inhibition of the binding by losartan in the left ventricles of hearts from LNSV- and LNSV–AT1R-AS–treated SHR. While a dose-dependent inhibition of 125I-[Sar1,Ile8]-Ang II by losartan was observed in both groups, the level of binding in ventricles of LNSV–AT1R-AS–treated SHR was significantly lower than LNSV-treated SHR (Fig 6A). This decrease was the result of a 25% decrease in Bmax (47±3 fmol/mg in LNSV–AT1R-AS treatment versus 63±4 fmol/mg in LNSV treatment) rather than Kd (0.3±0.04 nmol/L in LNSV–AT1R-AS treatment versus 0.35±0.06 nmol/L in LNSV treatment) (Fig 6B). The effect of LNSV–AT1R-AS treatment on Ang II receptors in adrenal glands was determined with the use of autoradiography. The rationale for using this technique was the limited size of the tissue and to differentiate the effects on AT1 and AT2 receptors. Binding of 125I-[Sar1,Ile8]-Ang II to the adrenal cortical region of saline-, LNSV-, and LNSV–AT1R-AS–treated SHR was displaced by losartan (Fig 7iii), whereas in the medulla it was displaced by PD123319 (Fig 7ii). This finding confirms a previously reported distribution of AT1 and AT2 receptors in the adrenal gland.32 LNSV treatment showed no significant changes in the total (Fig 7i), PD123319- (Fig 7iii), or losartan- (Fig 7ii) displaceable 125I-[Sar1,Ile8]-Ang II binding compared with saline-treated SHR. In contrast, LNSV–AT1R-AS treatment caused a substantial decrease in the losartan-displaceable 125I-[Sar1,Ile8]-Ang II binding in the cortical region (Fig 7i, ii). However, PD123319-displaceable binding in the medullary region did not change (Fig 7i, iii). Quantitation of cortical binding revealed approximately a 40% decrease in Bmax for 125I-[Sar1,Ile8]-Ang II binding. The indirect BPs for the 4-month LNSV-SHR used for measurement of the AT1R were higher than for the age-matched LNSV–AT1R-AS–treated SHR (167±9 mm Hg versus 138±2 mm Hg).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The results of this study establish that the viral-mediated delivery of AT1R-AS in developing rats results in a long-term attenuation of high BP. This reduction in BP was selectively manifested in SHR and not in control WKY. In contrast to selectivity of AT1R-AS in lowering the basal BP in SHR, the pressor and dipsogenic responses to Ang II were reduced in AT1R-AS treatment in both WKY and SHR. The reduction in BP achieved by AT1R-AS treatment was similar to that observed with losartan treatment. In addition, losartan treatment in AT1R-AS–treated SHR produced no further fall in BP. Collectively, these data suggest that in the developing SHR, interruption of AT1R expression with this gene transfer technique prevents the development of high BP in a manner similar to losartan treatment, except that this technology provides a significantly more prolonged antihypertensive effect.
There are numerous reports suggesting that the RAS is important for the development and maintenance of high BP in SHR.11 15 33 34 Additionally, it has been hypothesized that there is a critical period during the development of hypertension in SHR in which the RAS is essential and that interruption of the RAS activity during this period results in a prolonged antihypertensive effect even after the cessation of therapy.9 10 11 Our results support this concept in that the use of LNSV–AT1R-AS prevented the development of hypertension in SHR when administered to 5-day-old animals. In addition, it is likely that a continuous presence of AT1R-AS during early development may produce changes in Ang II target cells to reduce AT1R function by some as yet unidentified mechanism. At this time we do not know the duration of this antihypertensive effect; however, our data demonstrate that the long-lasting effect is maintained for at least 90 days after a single treatment.
Administration of the LNSV–AT1R-AS resulted in a rapid incorporation of AT1R-AS into the tissues, as demonstrated by the expression of this transcript within 3 days. In fact, it is possible, on the basis of our in vitro experiments, that this expression can occur even earlier. Infection of brain cells and vascular smooth muscle cells with LNSV–AT1R-AS results in the expression of AT1R-AS within 12 hours (Lu and Raizada, unpublished data, 1997). Although the transcript for AT1R-AS is present early and continues to be expressed by 30 days, this expression decreases with time and is no longer apparent by 60 days posttreatment. Despite the lack of expression, AT1R function is still compromised for up to 90 days posttreatment. That is, 3 months after a single treatment of LNSV–AT1R-AS, BP was significantly reduced in SHR compared with their saline- or LNSV-treated controls, and BP was not significantly different from that observed in WKY. We have used LNSV, the viral carrier, as control, since it matches everything from LNSV–AT1R-AS except AT1R-AS. Our results, therefore, suggest that a continued expression of AT1R-AS is not required for production of the antihypertensive effects and support the concept that there is a critical stage in the development of hypertension in SHR.8 9 10 11 However, the fact that AT1R-mediated responses are significantly depressed even though the AT1R-AS transcript is no longer present would suggest that some type of more prolonged antagonistic effect is maintained. For example, AT1R-AS expression at an early developmental stage may downregulate the native gene expression of the AT1R in various tissues (heart and adrenal). As a result, the AT1R-mediated high BP in SHR is attenuated.
The antihypertensive effects of LNSV–AT1R-AS treatment are specific and AT1R mediated: (1) treatment with the viral carrier LNSV had no effect on any of the parameters, such as body weight or food and water intake, in either of the strains; (2) losartan treatment caused no further decrease in BP in the LNSV–AT1R-AS–treated SHR; (3) the magnitude of the fall in BP with losartan treatment was similar to that observed with AT1R-AS treatment; (4) construction of the LNSV vector was such that the virus is deficient in expression of any viral protein that would essentially eliminate the nonspecificity of the viral particles16 ; and (5) the AT2 receptor and its mRNA levels were not affected in adrenal glands of LNSV–AT1R-AS–treated WKY and SHR compared with LNSV-treated controls.35 This observation is significant in view of the fact that Ang II binds to both AT1 and AT2 receptor subtypes with relatively similar affinity and both subtypes are G-protein–coupled receptors and have significant degrees of structural homology.36 37
Although AT1R function was reduced with AT1R-AS treatment in WKY, there was no effect on BP. This result would strongly suggest that in control animals the RAS is probably not important in normal BP regulation, which is consistent with previous reports.15 However, it is clear from the data obtained from SHR that the RAS is definitely involved in the pathogenesis of BP regulation in the hypertensive state.
Using two in vivo techniques to assess AT1R function demonstrated that LNSV–AT1R-AS treatment significantly reduced both the dipsogenic and pressor responses in both SHR and WKY. The EC50 data for the pressor responses to Ang II suggest that the sensitivity to Ang II was not altered in the LNSV–AT1R-AS–treated animals. Coupled with the depressed responses to Ang II, the data suggest that receptor availability is reduced with LNSV–AT1R-AS treatment. This is consistent with our observation of a decrease in AT1R in adrenals of 100-day SHR treated with LNSV–AT1R-AS. Likewise, the binding studies demonstrate a decrease in cardiac AT1R numbers after 4 months of treatment with LNSV–AT1R-AS. Measurement of AT1R at an earlier time period, especially when the functional changes are first observed, must be carried out to further support the observation that receptor function is reduced earlier in this treatment paradigm. From our data, we would suggest that the expression of AT1R-AS occurs early and results in some translational impairment of AT1R. This impairment apparently leads to a decrease in AT1R number and function. At this time we cannot definitively state whether AT1R gene expression is decreased on a chronic basis or if there is some developmental expression of other transcription factor(s) that may be responsible for the continued reduction in AT1R function even though the AT1R-AS transcript is no longer expressed. Likewise, it is possible that the antisense treatment may have long-lasting effects on various tissues that are important in BP regulation, such as the vasculature in altering peripheral resistance, the adrenals in altering catecholamine secretion, or the kidney in altering salt and water balance. These are but a few areas of future study.
An interesting question arises from these studies concerning the accessibility of LNSV–AT1R-AS to the brain and the role of brain RAS in the control of BP in this treatment. This is particularly important in view of the demonstrated role of brain Ang II in the control of hypertension in SHR19 20 21 38 and the fact that intracerebroventricular injections of antisense oligonucleotides to angiotensinogen or AT1R selectively lower BP in this animal model.19 20 21 Our present data suggest that brain AT1R may be involved. This supposition is based on the observation that Ang II–induced dipsogenic response was significantly attenuated in LNSV–AT1R-AS–treated rats. However, further experiments to demonstrate the expression of AT1R-AS, coupled with intracerebroventricularly injected Ang II–mediated BP responses, would be needed to support this view, especially in light of recent studies demonstrating that antisense oligonucleotides to angiotensinogen lower BP in SHR when administered systemically.22 23
Losartan treatment is widely accepted to be a standard therapy in the control of Ang II–dependent hypertension. We compared BP-lowering effects of LNSV–AT1R-AS treatment with those of losartan to determine the efficacy of gene therapy. Administration of losartan produces a similar and comparable reduction of BP in SHR as does the LNSV–AT1R-AS treatment. Additionally, Ang II–induced BP response also is attenuated by both treatments in SHR and WKY. In spite of these important similarities, there are differences between the two treatments. First, the BP-lowering effect of losartan is maximal 2 to 4 hours after its administration and is nearly normalized by 24 hours. In contrast, LNSV–AT1R-AS treatment lasts for 90 days, the duration of this study. Second, 4 hours post–losartan treatment there is a 34-fold and 65-fold increase in plasma Ang II levels in WKY and SHR, respectively. This observation is consistent with our previous study in which chronic losartan treatment has been shown to increase plasma Ang II levels.26 In contrast, treatment of rats with LNSV–AT1R-AS shows no significant increase in plasma Ang II levels. This is an important finding in view of the concerns raised for losartan therapy as to the possible implications of increased Ang II levels in hypertensives. Thus, these observations show that gene therapy with AT1R-AS has prolonged antihypertensive effects without the possible adverse effects of elevated plasma Ang II.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This work was supported by grants from NIH (HL56921) and the American Heart Association, Florida affiliate. LNSV was kindly provided by Geoffrey Owens, University of Colorado Health Science Center. We thank Dr Ronald Smith of DuPont-Merck (Wilmington, Del) for the generous supply of losartan potassium and Jennifer Brock for typing and preparation of this manuscript.
Received January 20, 1997; first decision February 14, 1997; accepted February 14, 1997.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Goldfarb DA. Altered vascular reactivity and baroreflex. Ren Vasc Dis Transplant. 1994;21:187-194.
2. Gifford RW. Approach to treatment of hypertension. In: Gifford RW, ed. Hypertension Primer: The Essential High Blood Pressure. Dallas, Tex: American Heart Association; 1993:285-288.
3. Berecek KH, Kirk KA, Nagahama S, Oparil S. Alterations in sympathetic function in spontaneously hypertensive rats after chronic administration of captopril. Am J Physiol. 1987;252:H796-H806.[Medline] [Order article via Infotrieve]
4. MacGregor GA. Blood pressure, angiotensin-converting enzyme inhibitors and the kidney. Am J Med. 1992;92:205-275.
5. Kang PM, Landau AJ, Eberhardt RT, Frishman WH. Angiotensin II receptor antagonists: a new approach to blockade of the renin-angiotensin system. Am Heart J. 1994;127:1388-1401.[Medline] [Order article via Infotrieve]
6. Van Zwieten PA. Development and trends in the drug treatment of essential hypertension. J Hypertens. 1992;10:S1-S12.
7. Goldberg MR, Bradstreet TE, McWilliams EJ, Tanaka WK,
Lipert S, Bjornsson TD, Waldman SA, Osborne B, Pivadori L, Lewis
G. Biochemical effects of losartan, a nonpeptide
angiotensin II receptor antagonist, on the
renin-angiotensin-aldosterone system in
hypertensive patients. Hypertension. 1995;25:37-46.
8. Berecek KH, Zhang L. Biochemistry and cell biology of angiotensin-converting enzyme and converting enzyme inhibitors. In: Mukhopadhyay AK, Raizada MK, eds. Tissue Renin-Angiotensin Systems. New York, NY: Plenum Press; 1995:141-168.
9. Harrap SB, Van der Merwe WM, Griffin SA, Macpherson F,
Lever AF. Brief angiotensin-converting enzyme
inhibitor treatment in young spontaneously hypertensive
rats reduces blood pressure long term. Hypertension. 1990;16:603-614.
10. Wu J-N, Berecek KH. Prevention of genetic
hypertension by early treatment of spontaneously hypertensive rats with
the angiotensin-converting enzyme inhibitor
captopril. Hypertension. 1993;22:139-146.
11. Berecek KH, King SJ, Wu J-N. Angiotensin-converting enzyme and converting enzyme inhibitors. In: Raizada MK, Phillips MI, Sumners C, eds. Cellular and Molecular Biology of the Renin-Angiotensin System. Boca Raton, Fla: CRC Press; 1993:183-220.
12. Burnier M, Waeber B, Brunner HR. The advantages of angiotensin II antagonism. J Hypertens Suppl. 1994;12:S7-S15.
13. Bonnardeaux A, Davies E, Jeunemaitre X, Fery I, Charru
A, Clauser E, Tiret L, Combien F, Corvol P, Soubrier F.
Angiotensin II type 1 receptor gene polymorphisms in
human essential hypertension. Hypertension. 1994;24:63-69.
14. Carr AA, Prisant LM. Losartan: first of a new class of angiotensin antagonists for the management of hypertension. J Clin Pharmacol. 1996;36:3-12.[Abstract]
15. Timmermans PBMWM, Wong PC, Chiu AT, Herblin WF, Benfield P, Carini DJ, Lee RJ, Wexler RR, Saye JAM, Smith RD. Angiotensin II receptor and angiotensin II receptor antagonist. Pharmacol Rev. 1993;45:205-251.[Medline] [Order article via Infotrieve]
16. Rolfs A, Weber-Rolfs I, Regitz-Zagrosek V, Kallisch H, Riedel K, Fleck E. Genetic polymorphisms of the angiotensin II type I (AT1) receptor gene. Eur Heart J. 1994;15(suppl D):108-112.
17. Lu D, Yu K, Raizada MK. Retrovirus-mediated
transfer of an angiotensin type I receptor
(AT1R) antisense sequence decreases AT1Rs and
angiotensin II action in astroglial and neuronal cells in
primary cultures from the brain. Proc Natl Acad Sci
U S A. 1995;92:1162-1166.
18. Lu D, Raizada MK. Delivery of
angiotensin II type 1 receptor antisense inhibits
angiotensin action in neurons from hypertensive rat
brain. Proc Natl Acad Sci U S A. 1995;92:2914-2918.
19. Wielbo D, Sernia C, Gyurko R, Phillips MI.
Antisense inhibition of hypertension in the spontaneously hypertensive
rat. Hypertension. 1995;25:314-319.
20. Gyurko R, Wielbo D, Phillips MI. Antisense inhibition of AT1 receptor mRNA and angiotensinogen mRNA in the brains of spontaneously hypertensive rats reduces hypertension of neurogenic origin. Regul Pept. 1993;49:167-174.[Medline] [Order article via Infotrieve]
21. Phillips MK, Wielbo D, Gyurko R. Antisense inhibition of hypertension: a new strategy for renin-angiotensin candidate genes. Kidney Int. 1994;46:1554-1556.[Medline] [Order article via Infotrieve]
22. Wielbo D, Simm A, Phillips MI, Toffolo S.
Inhibition of hypertension by peripheral administration of
antisense oligodeoxynucleotides.
Hypertension. 1996;28:147-151.
23. Tomita N, Morishita R, Higaki J, Kaneda Y, Mikami H,
Ogihara T. Transient decrease in high blood pressure by in vivo
transfer of antisense oligonucleotide agonist rat
angiotensinogen. Hypertension. 1995;26:131-136.
24. Lu D, Yu K, Paddy MR, Rowland NE, Raizada MK. Regulation of norepinephrine transporter system by angiotensin II in neuronal cultures of normotensive and spontaneously hypertensive rat brains. Endocrinology. 1996;137:763-772.[Abstract]
25. Iyer SN, Katovich MJ. Effect of chronic losartan potassium treatment on fructose-induced hypertension. Life Sci. 1994;55:139-144.
26. Iyer SN, Wright BE, Strubbes G, Hanley K, Katovich MJ. Chronic losartan treatment blocks isoproterenol-induced dipsogenesis. Physiol Behav. 1995;58:283-286.[Medline] [Order article via Infotrieve]
27. Iyer SN, Katovich MJ. Vascular reactivity to phenylephrine and angiotensin II in hypertensive rats associated with insulin resistance. Clin Exp Hypertens. 1996;18:227-242.[Medline] [Order article via Infotrieve]
28. Song K, Kurobe Y, Kanehara H, Okunishi H, Wada T, Inada Y, Nishikawa K, Miyazaki M. Quantitative localization of angiotensin II receptor subtypes in spontaneously hypertensive rats. Blood Press. 1994;5(suppl):21-26.
29. Baker KM, Campanile CP, Trachte GJ, Peach MJ. Identification and characterization of rabbit angiotensin II myocardial receptor. Circ Res. 1994;54:286-293.
30. Raizada MK, Lu D, Tang W, Kurian P, Sumners C.
Increased angiotensin II type 1 receptor gene expression in
neuronal cultures from spontaneously hypertensive rats.
Endocrinology. 1993;132:1715-1722.
31. Yang H, Lu D, Raizada MK. Lack of cross-talk
between 
1-adrenergic and angiotensin type 1
receptors in neurons of spontaneously hypertensive rat brain.
Hypertension. 1996;27:1277-1283.
32. Balla T, Baukal AJ, Eng S, Catt KJ. Angiotensin II receptor subtypes and biological responses in the adrenal cortex and medulla. Mol Pharmacol. 1991;40:401-406.[Abstract]
33. Bachmann J, Wagner J, Haufe C, Wystrychowski A, Ciechanowiez A, Ganten D. Modulation of blood pressure and the renin-angiotensin system in transgenic and spontaneously hypertensive rats after ovariectomy. J Hypertens Suppl. 1993;11:S226-S227.[Medline] [Order article via Infotrieve]
34. Kawano Y, Yoshida K, Matsuoka H, Omae T. Chronic effects of central and systemic administration of losartan on blood pressure and baroreceptor reflex in spontaneously hypertensive rats. Am J Hypertens. 1994;7:536-542.[Medline] [Order article via Infotrieve]
35. Iyer SN, Lu D, Katovich MJ, Raizada MK. Retroviral-mediated delivery of antisense cDNA of AT1 receptor attenuates development of high blood pressure in the spontaneously hypertensive rat. In: Program and abstracts of the 10th International Congress of Endocrinology; 1996:105; San Francisco, Calif. Abstract OR32-1.
36. Mukoyama M, Nakajima M, Horiuchi M, Sasamura H, Pratt
RE, Dzau VJ. Expression cloning of type 2
angiotensin II receptor reveals a unique class of
seven-transmembrane receptors. J Biol Chem. 1993;268:24539-24542.
37. Timmermans PBMWM, Benfield P, Chiu AT, Herblin WF, Wong PC, Smith RD. Angiotensin II receptors and functional correlate. Am J Hypertens. 1992;5:2215-2355.
38. Phillips MI. Functions of angiotensin in the central nervous system. Annu Rev Physiol. 1987;49:413-435.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  J. L. Grobe, A. P. Mecca, H. Mao, and M. J. Katovich Chronic angiotensin-(1-7) prevents cardiac fibrosis in DOCA-salt model of hypertension Am J Physiol Heart Circ Physiol, June 1, 2006; 290(6): H2417 - H2423. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. K. Raizada and S. D. Sarkissian Potential of Gene Therapy Strategy for the Treatment of Hypertension Hypertension, January 1, 2006; 47(1): 6 - 9. [Full Text] [PDF]
|
|
|
|
 |
|
 M. J Huentelman, J. L Grobe, J. Vazquez, J. M Stewart, A. P Mecca, M. J Katovich, C. M Ferrario, and M. K Raizada Protection from angiotensin II-induced cardiac hypertrophy and fibrosis by systemic lentiviral delivery of ACE2 in rats Exp Physiol, September 1, 2005; 90(5): 783 - 790. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Katovich, J. L. Grobe, M. Huentelman, and M. K. Raizada Angiotensin-converting enzyme 2 as a novel target for gene therapy for hypertension Exp Physiol, May 1, 2005; 90(3): 299 - 305. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Wang, S. Gallinat, H.-w. Li, C. Sumners, M. K. Raizada, and M. J. Katovich Elevated blood pressure in normotensive rats produced by 'knockdown' of the angiotensin type 2 receptor Exp Physiol, May 1, 2004; 89(3): 313 - 322. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Yang, P. Y. Reaves, M. J. Katovich, and M. K. Raizada Decrease in Hypothalamic Gamma Adducin in Rat Models of Hypertension Hypertension, February 1, 2004; 43(2): 324 - 328. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Pachori, M. J. Huentelman, S. C. Francis, C. H. Gelband, M. J. Katovich, and M. K. Raizada The Future of Hypertension Therapy: Sense, Antisense, or Nonsense? Hypertension, February 1, 2001; 37(2): 357 - 364. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 A. S. Pachori, H. Wang, C. H. Gelband, C. M. Ferrario, M. J. Katovich, and M. K. Raizada Inability to Induce Hypertension in Normotensive Rat Expressing AT1 Receptor Antisense Circ. Res., June 9, 2000; 86(11): 1167 - 1172. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Wang, P. Y. Reaves, M. L. Gardon, K. Keene, D. S. Goldberg, C. H. Gelband, M. J. Katovich, and M. K. Raizada Angiotensin I-Converting Enzyme Antisense Gene Therapy Causes Permanent Antihypertensive Effects in the SHR Hypertension, January 1, 2000; 35(1): 202 - 208. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. H. Gelband, H. Wang, M. L. Gardon, K. Keene, D. S. Goldberg, P. Y. Reaves, M. J. Katovich, and M. K. Raizada Angiotensin I-Converting Enzyme Antisense Prevents Altered Renal Vascular Reactivity, but Not High Blood Pressure, in Spontaneously Hypertensive Rats Hypertension, January 1, 2000; 35(1): 209 - 213. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Y. Reaves, C. H. Gelband, H. Wang, H. Yang, D. Lu, K. H. Berecek, M. J. Katovich, and M. K. Raizada Permanent Cardiovascular Protection From Hypertension by the AT1 Receptor Antisense Gene Therapy in Hypertensive Rat Offspring Circ. Res., November 12, 1999; 85 (10): e44 - e50. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. G. Luke Hypertensive nephrosclerosis: pathogenesis and prevalence : Essential hypertension is an important cause of end-stage renal disease Nephrol. Dial. Transplant., October 1, 1999; 14(10): 2271 - 2278. [Full Text] [PDF]
|
|
|
|
 |
|
 J. Zicha and J. Kunes Ontogenetic Aspects of Hypertension Development: Analysis in the Rat Physiol Rev, October 1, 1999; 79(4): 1227 - 1282. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Wang, M. J. Katovich, C. H. Gelband, P. Y. Reaves, M. I. Phillips, and M. K. Raizada Sustained Inhibition of Angiotensin I-Converting Enzyme (ACE) Expression and Long-Term Antihypertensive Action by Virally Mediated Delivery of ACE Antisense cDNA Circ. Res., October 1, 1999; 85(7): 614 - 622. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Katovich, C. H. Gelband, P. Reaves, H.-W. Wang, and M. K. Raizada Reversal of hypertension by angiotensin II type 1 receptor antisense gene therapy in the adult SHR Am J Physiol Heart Circ Physiol, September 1, 1999; 277(3): H1260 - H1264. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
C. H. Gelband, P. Y. Reaves, J. Evans, H. Wang, M. J. Katovich, and M. K. Raizada Angiotensin II Type 1 Receptor Antisense Gene Therapy Prevents Altered Renal Vascular Calcium Homeostasis in Hypertension Hypertension, January 1, 1999; 33(1): 360 - 365. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. Martens, P. Y. Reaves, D. Lu, M. J. Katovich, K. H. Berecek, S. P. Bishop, M. K. Raizada, and C. H. Gelband Prevention of renovascular and cardiac pathophysiological changes in hypertension by angiotensin II type 1 receptor antisense gene therapy PNAS, March 3, 1998; 95(5): 2664 - 2669. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||