© 1999 American Heart Association, Inc.
UltraRapid Communications |
Permanent Cardiovascular Protection From Hypertension by the AT1 Receptor Antisense Gene Therapy in Hypertensive Rat Offspring
From the Department of Physiology (P.Y.R., C.H.G., H.W., H.Y., D.L., M.K.R.), College of Medicine, Department of Pharmacodynamics (M.J.K.), College of Pharmacy, University of Florida, Gainesville, Fla; Department of Physiology and Biophysics (K.H.B.), University of Alabama, Birmingham, Ala.
Correspondence to Mohan K. Raizada, PhD, Professor, Department of Physiology, College of Medicine, University of Florida, PO Box 100274, Gainesville, FL 32610. E-mail mraizada{at}phys.med.ufl.edu
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Abstract |
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Abstract—Our previous studies have demonstrated that the introduction of angiotensin II type I receptor antisense (AT1R-AS) cDNA by a retrovirally mediated delivery system prevents the development of hypertension in the spontaneously hypertensive rat (SHR), an animal model for primary hypertension in humans. These results have led us to propose the hypothesis that an interruption of the renin-angiotensin system (RAS) activity at a genetic level would prevent hypertension on a permanent basis. F1 and F2 generations of offspring from a retroviral vector, LNSV- and LNSV-AT1R-AS–treated SHR, were generated, and various physiological parameters indicative of hypertension were studied and compared with those of their parents to investigate this hypothesis. Both F1 and F2 generations of LNSV-AT1R-AS–treated SHR expressed a persistently lower blood pressure, decreased cardiac hypertrophy and fibrosis, decreased medial thickness, and normalization of renal artery excitation-contraction coupling, Ca2+ current, and [Ca2+]i when compared with offspring derived from the LNSV-treated SHR. In fact, the magnitude of the prevention of these pathophysiological alterations was similar to that observed in the LNSV-AT1R-AS–treated SHR parent. The prevention of cardiovascular pathophysiology and expression of normotensive phenotypes are, at least in part, a result of integration and subsequent transmission of AT1R-AS from the SHR parents to offspring. These data demonstrate that a single intracardiac injection of LNSV-AT1R-AS causes a permanent cardiovascular protection against hypertension as a result of a genomic integration and germ line transmission of the AT1R-AS in the SHR offspring. The full text of this article is available at http://www.circresaha.org.
Key Words: AT1 receptor antisense • gene therapy • hypertension • SHR • antisense transmission to offspring
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Introduction |
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Hypertension is a complex disease that is associated with major economic and emotional burdens to society. It is also a significant risk factor in stroke, arteriosclerosis, heart failure, coronary artery disease, and progressive renal damage.1 2 Decades of investigation have established that a hyperactive renin-angiotensin system (RAS) is one of the many physiological events that becomes dysfunctional in hypertension.2 3 4 5 This fact is further supported by the observation that pharmacological interruption in the activity of the RAS has proved to be highly successful in the treatment and management of hypertension in a significant population of hypertensive patients.6 7 In spite of this success, the traditional pharmacological therapy targeted to inhibit specific components of the RAS suffers from many significant disadvantages. One is patient compliance. This is particularly important in view of the fact that chronic administration of drugs is almost always necessary for a persistent, long-term antihypertensive effect, and prolonged therapy with certain antihypertensive agents can lead to significant side effects such as coughing, angioedema, hypotension, renal dysfunction, and hyperkalemia.8 9 10 Additionally, although a traditional pharmacological strategy can be successful in the control and the management of high blood pressure (BP), its effectiveness in the prevention and/or reversal of other associated pathophysiological alterations such as tissue remodeling leading to end-organ damage remains to be proven.11 In fact, disproportionate reversal of some alterations, such as left ventricular hypertrophy and peripheral resistance, has proven to be unfavorable in the successful management of this disease.12 13
In view of the success of pharmacological agents targeted toward the inhibition of the RAS, and the recent rapid advances in gene delivery, we decided to investigate whether antisense gene therapy could be a superior treatment for hypertension. We used a retrovirally mediated delivery system to administer AT1 receptor antisense (AT1R-AS) cDNA in vitro.14 15 These studies established that AT1R-AS cDNA could be incorporated into the genome and that the transcript could be expressed on a long-term basis. This expression was associated with a significant alteration of the AT1R-mediated cellular action of angiotensin II (Ang II) indicating that such an approach was feasible. Animal experiments were highly successful and demonstrated that intracardiac delivery of AT1R-AS in the neonatal spontaneously hypertensive rat (SHR) prevented the development of hypertension, renal, and cardiovascular pathophysiological changes on a long-term basis.16 17 18 The antihypertensive effect was associated with a robust long-term expression of AT1R-AS transcript. These studies led us to hypothesize that the interruption in the activity of the RAS during development by the AT1R-AS would attenuate hypertension on a permanent basis. The present study was designed to support or refute this hypothesis.
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Materials and Methods |
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Preparation of Viral Particles Containing AT1R-AS
AT1R-AS was cloned in a retroviral vector containing long terminal repeats, neomycin selection, and simian virus promoter (LNSV) as described previously.14 15 19 Media-containing viral particles from the PA317 cells were collected and concentrated to provide 1x109 to 2x1010 cfu/mL as described previously.19 Viral particles that did not contain AT1R-AS (LNSV) were also prepared by the same protocol and used in control experiments.
Administration of LNSV- and LNSV-AT1R-AS–Containing
Viral Particles in Rats
Five-day-old Wistar Kyoto (WKY) and spontaneously hypertensive
(SHR) rats were divided into two groups: virus-along control (LNSV) or
virus-containing AT1R-AS
(LNSV-AT1R-AS). Animals were injected with a
bolus of 1x1010 cfu of viral particles in 10
µL physiological saline intracardially, weaned,
and raised as described previously.16 17 18 Indirect BP was
monitored throughout development. Two sets of parents of LNSV- or
LNSV-AT1R-AS–treated WKY and SHR were bred at
100 days of age to generate F1 offspring. Similar
to their parents, they were weaned, raised, and monitored for BP
indirectly by the tail-cuff method.16 17 18 At 100 days of
age, two sets of F1 offspring of LNSV- and
LNSV-AT1R-AS–treated WKY and SHR were bred to
generate F2 offspring. One hundred-day-old
parents and F1 and F2
offspring were used for all biochemical and
physiological experiments.
Biochemical Experiments
Binding of 125I-Sar1
1le8-Ang II to membrane AT1
receptors was carried out as described elsewhere.14 15
Computer-assisted Scatchard analysis was done to determine the
Bmax and Kd values.
Polymerase chain reaction (PCR) followed by a Southern analysis
was carried out to determine the genomic integration of the
AT1R-AS essentially as described
elsewhere.20 21 22 The expression of
AT1R-AS transcript in various Ang II target
tissues was carried out by a semiquantitative reverse transcriptase
(RT)-PCR method as we described previously.16
Physiological Protocols
Indirect BP was monitored in nonanesthetized animals by
a standard tail-cuff method.16 17 18 Direct BP was
determined in free-moving animals as previously
described.16 17 18 For vascular smooth muscle studies,
3-mm-long segments of rat renal resistance arterioles were
used.18 23 Contractile responses to KCl,
phenylephrine, and acetylcholine were evaluated as
described previously.18
[Ca2+]i in dissociated
renal resistance arterial cells in response to KCl and Ang
II was measured exactly as we described previously.23
Ca2+ current recordings were carried out
by voltage-clamping single cells.23
Pathophysiological Parameters
Heart weight–to–body weight ratio, cardiac fibrosis, and
collagen volume in both endocardium and epicardium were determined by
our previously published protocols.18 Animals were killed
and perfused with fixative without applying additional pressure,
according to a previously published protocol.24
Morphometric analysis of thoracic aorta to determine the wall
thickness and media/lumen ratio was carried out by an established
protocol.24
Statistics
Results are expressed as mean±SE. Statistical significance was
evaluated with repeated measures, ANOVA, and Student’s t
test for unpaired data. Differences were considered significant at
P<0.05. For vascular studies, all rings were normalized to
tissue weights and cross-sectional area.
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Results |
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Five-day-old WKY rats and SHR were administered 1x109 cfu of LNSV (control virus) or LNSV-AT1R-AS (LNSV virus containing AT1R-AS cDNA) via an intracardiac injection. Animals were allowed to grow and were subjected to routine indirect BP monitoring. Two sets of LNSV-treated WKY rats (mean BP 129±6.0 mm Hg) and SHR (mean BP 198±18 mm Hg) and LNSV-AT1R-AS–treated WKY and SHR (mean BP 120±6.0 and 153±9 mm Hg, respectively) were used as parents for breeding and an F1 generation of rats was produced. Offspring from LNSV-AT1R-AS–treated SHR expressed significantly lower BP as early as 80 days of age compared with their LNSV-treated SHR control. By 120 days, the average BP of the AT1R-AS–treated SHR offspring was of 45±9 mm Hg lower than that observed in the LNSV-treated SHR offspring (150±10 versus 185±11 mm Hg, Figure 1
). No significant effect on BP was
observed between offspring of AT1R-AS– and
LNSV-treated WKY rats. The LNSV-AT1R-AS–treated
WKY rat group was not used further in our experiment because the
parents showed no effect on BP and cardiovascular and
renal pathophysiology18 and the BP of the offspring showed
no change. The F2 generation was produced by
mating two pairs of F1 SHR whose parents were
treated at 5 days of age with LNSV alone or
LNSV-AT1R-AS. As observed for the
F1 generation, the F2
offspring derived from the original
AT1R-AS–treated SHR showed a significantly lower
BP compared with F2 offspring derived from
LNSV-treated SHR controls. At 120 days of age, F2
SHR derived from the AT1R-AS–treated rats had an
average BP of 135±12 mm Hg versus 165±10 mm Hg in
F2 offspring from LNSV-treated SHR (Figure 1). Genomic DNA from various tissues of
LNSV-AT1R-AS–treated SHR parents and their
F1 and F2 offspring was
isolated. It was subjected to PCR to amplify
LNSV-AT1R-AS and followed by the Southern
analysis to detect the AT1R-AS gene
incorporation. All of the tissues analyzed from the parents and
both generations of offspring showed integration of
LNSV-AT1R-AS (Figure 2A). In addition, the expression of
AT1R-AS in both parents and offspring was evident
(Figure 2B).
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1 kb) was randomly labeled with
[32P]-dATP and used for hybridization by the standard
protocol.
The effect of losartan, an AT1
receptor–specific antagonist and well-established
antihypertensive drug, on offspring derived from
AT1R-AS–treated SHR was also studied. Treatment
of control SHR by losartan resulted in a 29±6 mm Hg
decrease in BP (Figure 3). A comparable
decrease in BP was observed in LNSV-treated SHR. In contrast, no
significant decrease in BP was observed in the LNSV-
AT1R-AS–treated SHR. Similar to
AT1R-AS–treated parents, their
F1 and F2 offspring showed
little lowering of BP by losartan whereas offspring from
LNSV-treated parents experienced a 25 to 29 mm Hg decrease in BP
(Figure 3). These observations confirm that the
AT1R-AS treatment of parents produced
antihypertensive effects in both parents and offspring through an
AT1 receptor–mediated mechanism and that the
antihypertensive effect is as effective as the
AT1 receptor antagonist therapy. The
conclusion that antisense gene therapy influences BP in the SHR by
affecting the levels of AT1 receptors is
demonstrated by comparing the cardiac AT1
receptors in the F1 generation of LNSV- and
AT1R-AS–treated SHR. Total numbers of
AT1 receptors (Bmax) in the
ventricles of the F1 offspring derived from
AT1R-AS–treated SHR were decreased by 36%
compared with offspring from parents of LNSV-treated SHR (Table 1).
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Tissue remodeling and associated ultrastructural changes in tissues
relevant to cardiovascular functions such as heart,
kidney, and arteries are major contributing factors in the morbidity
and mortality associated with hypertension. For example, left
ventricular hypertrophy, a compensatory
response of the heart to an increase in peripheral vascular
resistance is an important pathophysiological
manifestation of hypertension. We determined whether
AT1R-AS treatment influences cardiac
pathophysiology, and, if so, could this effect be maintained in their
offspring. Heart weights of untreated SHR were 68% higher than those
of WKY rats (Table 1
). AT1R-AS treatment
significantly prevented this cardiac hypertrophy in
parents.18 Similarly, heart weights of the
F1 generation of
AT1R-AS–treated SHR were 26% lower than the
F1 generation of LNSV-treated SHR (Table 1). Cardiac hypertrophy was significantly prevented
in the F2 generation of
AT1R-AS–treated SHR as well. Multifocal areas of
fibrosis in the myocardium are another characteristic of
hypertension in this model. Figure 4
provides an example of sections taken from the left
ventricular subendomyocardium of
F2 offspring derived from LNSV- and
AT1R-AS–treated SHR. Multiple areas of fibrosis
were clearly evident in the offspring of LNSV-treated SHR (Figure 4b) but were rarely observed in the offspring of
AT1R-AS–treated animals (Figure 4c) or in
the control WKY rat (Figure 4a). Collagen volume in both
endocardium and epicardium, a measure of cardiac fibrosis, was <90%
decreased in the offspring of LNSV-AT1R-AS SHR
(Table 1), confirming the morphological detection of
fibrosis.
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Ultrastructural examination of the thoracic aorta of the offspring of
LNSV-AT1R-AS–treated SHR parents revealed a
significant decrease in the wall and medial thickness compared with
offspring of LNSV-treated SHR parents (Table 1). The lumen area
was increased in this group of SHR offspring. For example, wall
thickness in the SHR and LNSV-treated SHR offspring of
F2 generation was 36% to 90% greater than that
of the WKY control. F2 offspring from
LNSV-AT1R-AS–treated SHR demonstrated a 34%
decrease in wall thickness, which was closer to values for the same
measurement in WKY rats. Similarly, the media/lumen ratio was 42%
lower in this generation of LNSV-AT1R-AS–treated
SHR. These data clearly establish that AT1R-AS
treatment prevents these vascular
pathophysiological changes in this model of
hypertension.
We examined the pathophysiological changes in the
renal resistance arterioles and artery of the F1
and F2 offspring of
AT1R-AS–treated SHR parents. The rationale was
based on the fact that an increased vascular tone leading to an
increased renal vascular resistance is an important underlying
mechanism in the elevation of BP.25 26 The cellular
mechanisms responsible for this include an enhanced contractile
sensitivity to vasoactive agents, an impaired
endothelial-dependent vasorelaxation, increased
[Ca2+]i by its transport
across the vascular smooth muscle cell (VSMC) membrane, altered ion
channel activity in VSMC, and smooth muscle cell
hypertrophy and hyperplasia.23 We examined the
effect of parental treatment of AT1R-AS of the
SHR on the above pathophysiological
parameters in the renal resistance arteriole and renal
artery in the F1 and F2
offspring. Ultrastructural examination revealed that the thickness of
the intima and media and the overall arterial morphological
changes characteristic of hypertension were prevented in the SHR
offspring of AT1R-AS–treated parents (Figure 4d through 4f).
Next, we examined the effects of AT1R-AS
treatment on renal vascular reactivity. Our previous studies have shown
that the SHR renal arteriole expresses an enhanced contractile response
to KCl and phenylephrine. This enhancement, a result of a
leftward shift of the concentration-response curve reflecting in the
EC50 for KCl and phenylephrine, was
attenuated in AT1R-AS–treated
SHR.18 In the present study, the renal vascular
response in offspring of both F1 and
F2 generations of parents treated with
LNSV-AT1R-AS was examined. In the parents, the
vascular contractile responses to KCl and phenylephrine
were shifted rightward as a result of an increase in
EC50 values in both F1 and
F2 generation of offspring from
LNSV-AT1R-AS–treated SHR. Data for the
F2 generation are presented in Table 2 as an example. As a result, the
EC50 values for F1 and
F2 generations from the
LNSV-AT1R-AS–treated SHR parents were comparable
to those in the WKY rat. In contrast to the result with KCl and
phenylephrine when compared with WKY, the untreated and
LNSV-treated SHR showed a shift to the right in the
acetylcholine-induced vasorelaxation of precontracted renal arteriole
as reflected by an increase in the EC50 as well
as a decrease in the maximal effect. This effect was significantly
improved in the offspring of parents treated with
LNSV-AT1R-AS. For example, the
EC50 response of F2
offspring of LNSV-AT1R-AS–treated SHR was 77%
lower when compared with the LNSV-treated SHR and was similar to that
of the WKY rat. Similarly, the efficacy was 2.2-fold higher and
comparable to the WKY rat (Table 2). These data
demonstrate that endothelial dysfunction associated
with hypertension is prevented by AT1R-AS gene
therapy on a permanent basis.
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We also studied L-type Ca2+ current in
F1 and F2 generations from
LNSV- and LNSV-AT1R-AS–treated SHR parents. The
rationale for this experiment was based on our previous observations
that demonstrated that L-type Ca2+ current is
increased in VSMCs of the renal arterioles of the SHR.23
Ca2+ current was significantly decreased in both
F1 and F2 generations of
LNSV-AT1R-AS–treated SHR parents compared with
that from the LNSV-treated SHR parent. The mean I-V
relationship demonstrating this conclusion is presented in
Figure 5A for the
F2 generation of offspring. Differences in the
peak Ca2+ current are shown in Table 2.
Finally, we investigated the effects of KCl and Ang II on
[Ca2+]i in renal
arteriolar VSMCs. Our previous studies have established that KCl- and
Ang II–induced [Ca2+]i
were significantly elevated in the SHR compared with the WKY
rat.23 Data in Figure 5B show that KCl- or Ang
II–induced [Ca2+]i
increases in F1 offspring from
LNSV-AT1R-AS–treated SHR parents were
significantly attenuated when compared with the KCl and Ang II
responses in the offspring from LNSV-treated SHR parents. Similar data
were obtained in the F2 generation. These
findings provide additional evidence that alterations in the
[Ca2+]i homeostasis by
the SHR renal arteriolar cells are permanently prevented by
AT1R-AS gene therapy.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The observations presented in this study demonstrate that a single intracardial administration of a retroviral vector containing AT1R-AS causes a permanent protection against hypertension in this animal model of human primary hypertension. Thus, it suggests that an antisense strategy to inhibit the RAS at a genetic level is a conceptually novel approach for the prevention of hypertension. The reduction in BP is associated with the prevention of hypertension-associated pathophysiological and ultrastructural changes in the renal and cardiac tissue. In addition, this cardiovascular protection against hypertension is transmitted to at least two subsequent generations.
The most important question that arises from the study concerns the
mechanism of by which normotensive phenotypes are transmitted
from parents to offspring. Our data in Figure 2 support the
notion that the AT1R-AS is integrated into the
parental genome and is transmitted to the offspring. The proposed
germ-line transmission of the AT1R-AS is
consistent with previous reports demonstrating the integration
of retroviral vector and its germ-line transmission in other
systems.27 28 29 However, this study is unique because it
shows that the transmission and accompanied expression of the
AT1R-AS is associated with profound
antihypertensive physiological changes in the
offspring. Although we know little about the efficiency of this
transduction, it must be high enough to influence the expression of
antihypertensive phenotypes, an end point that is of ultimate
relevance to hypertension. The possibility that lack of a blood-gonadal
barrier and the presence of significant numbers of undifferentiated
germ cells in the neonatal rat cannot be ruled out. Thus, a critical
age of the rat at which the viral administration was carried out may be
the key for such a high efficiency of transduction in the
offspring.
In spite of our evidence in favor of AT1R-AS transmission, other possibilities to explain this prolonged antihypertensive effect should not be ruled out at the present time. For example, studies have shown that parental environment is critical in the development of hypertension.30 31 32 Thus, it is quite possible that the exposure of an antihypertensive environment by the AT1R-AS treatment of parents induces normotensive phenotype in the offspring. Cross studies with the SHR would support this review.32 33 In addition, the possibility that the AT1R-AS expression at a critical stage of SHR development may irreversibly prevent the parents and offspring from developing hypertension. This would be consistent with previous suggestions.34 Finally, it is also quite possible that a combination of these mechanisms may ultimately be responsible for such a dramatic protection against hypertension.
Finally, is antisense gene therapy that targets the RAS a therapeutic step forward? On the basis of our data, the answer has to be affirmative. In our model, a single injection of retroviral vector containing AT1R-AS offers permanent prevention of hypertension. It not only minimizes side effects but also resolves the compliance issue observed in traditional pharmacological therapy. However, caution must be taken in use of this vector for any long-term therapeutics because of some concern as to an unknown insertion site of the retroviral vector in the genome. Another caveat of this study is that its success depends on the identification of the genetic determinants of hypertension at the prehypertensive stage before the therapy can be considered for human use. Angiotensin I–converting enzyme (ACE) may be one such determinant. It is well-established that ACE gene polymorphism cosegregates with hypertension,35 36 and that mutations at key places in ACE are associated with the development of high BP.37 38 Thus, targeting ACE by such an antisense strategy may be important. Would gene therapy reverse hypertension in the adult animal? A pilot study demonstrates a relatively long-term reversal of high BP and other renal pathophysiological changes induced by hypertension in adult SHR.39 In conclusion, our observation provides an initial step forward toward the use of gene therapy for a permanent benefit to the cardiovascular system in the control of hypertension.
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Acknowledgments |
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This research was supported by grants from the National Institutes of Health (HL56921 and HL52189). Drs Phyllis Reaves and Hong Yang are postdoctoral fellows of the American Heart Association, Florida Affiliate. We thank Dr Craig Tisher, University of Florida, for his intellectual input throughout this study and his critical review of the manuscript. Technical support of Ling Liu and Rebecca Kochera and editorial help of Marya Fancey are gratefully acknowledged.
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Footnotes |
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1 These authors contributed equally to this study.
Received September 10, 1999; accepted October 8, 1999.
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