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Introduction
Epidemiological data have suggested a correlation
between dietary salt and blood pressure regulation in the
prevalence and progression of essential hypertension, although
the salt-blood pressure theory has remained the subject of
ongoing controversy in a number of
studies[1_4]. It has been demonstrated that the renin-angiotensin (RAS) is important
in the development and maintenance of hypertension in both
essential hypertensive patients and animal models of
hypertension[5,6]. Moreover, many studies have revealed that high
salt could stimulate the RAS and induce angiotensin II
receptor 1 (AT1R) overexpression, which may be responsible
for the elevation of blood
pressure[7,8]. The blockade or
interruption of RAS by pharmacological agents, such as the
angiotensin-converting enzyme inhibitor and
AT1R antago-nists, have been extremely successful in the treatment of
hypertension and the prevention of it complications. The
findings indicate that the RAS components are crucial for
the development of hypertension and cardiovascular
function.
Gene therapy is a promising strategy for the treatment of
hypertension[9]. Applications of antisense RAS genes
to block the expression of either angiotensinogen or
AT1R have demonstrated efficiency of this therapeutic strategy. Iyer
et al reported that retroviral vector-mediated antisense
AT1R cDNA gene delivery resulted in a chronic reduction up to
30_60 mmHg in hypertension in spontaneously
hypertensive rats (SHR) rather than Wistar Kyoto normotensive
rats[10]. The intracerebroventricular application of antisense
oligonucleotides, directed to either
AT1R or angiotensinogen mRNA also
significantly reduced blood pressure in
hypertensive animals with a single
injection[11_13]. Moreover, an injection of antisense cDNA or oligonucleotides of the
angiotensin-converting enzyme, renin, or angiotensinogen
genes also induced a reduction of blood pressure in
hypertensive animals[14_17]. All these findings suggest that
blocking RAS genes may serve as target or candidate genes for
hypertension gene therapy.
Another concern is which vector could be used to carry
genes for chronic or permanent reduction in blood pressure.
Several viral vectors, including
retroviruses, lentivirus virus, and recombinant adeno-associated virus vector (rAAV) can
be adaptive for gene therapy of genetic or chronic diseases.
However, the retrovirus vector should not be considered
due to the carcinogenic effects. rAAV, which either
integrates into the genome or remains in the nucleus as a stable
episome, offers the most attractive advantages and may be
the most promising gene vector for long-term
therapy[18,19]. The adeno-associated virus (AAV) is safe to use. It does
not induce any pathogenic responses and does not
replicate inside cells. The AAV is a defective parvovirus which
cannot replicate in cells without the presence of a wild-type
adenovirus[20,21]. The
AAV is effective as a vector as it
does not produce viral proteins that stimulate
inflammatory reactions and contains sufficient carrying
capacity for the insertion of an antisense cDNA for
AT1R. More recently, a new double-stranded rAAV was developed, and this double strand
can express more rapidly and efficiently than a single-strand
rAAV, which makes it more
attractive[22]. In the present study, the rAAV was used to carry the rat antisense
AT1R gene (rAAV-AT1-AS) for hypertension therapy. It was
demonstrated that the rat rAAV-AT1-AS delivery via intravenous
injection prevented the long-term, salt-induced development
of blood pressure and attenuated cardiovascular
complications of hypertension.
Materials and methods
Preparation of rAAV-AT1 -AS and rAAV-GFP (green
fluorescent protein, as control) The rAAV-D(+) vector
(double-stranded rAAV Vector plasma), the
adenovirus helper plasmid
pXX6, and the packaging plasmid
pXX2 have been described
previously[21]. A 660 bp fragment of rat vascular
AT1R cDNA (127_786) was amplified with RT-PCR from rat
kidney total RNA and ligated to the rAAV-D(+)
vector in the antisense orientation after being identified by sequencing.
The rAAV-AT1-AS and rAAV-GFP vector plasmids were
constructed by inserting the rat AT1 antisense cDNA and GFP
cDNA into the rAAV-D(+) vector driven by the
cytomegalovirus promoter at BamHI and NotI sites, respectively. The
vectors for rAAV-AT1-AS and rAAV-GFP were prepared
using a triple plasmid cotransfection method in 293 cell lines,
as described previously[21,23]. A total of 50 µg plasmid DNA
per 15 cm plate (rAAV-AT1-AS or rAAV-GFP vector plasmids
/pXX2/pXX6 were 1:1:1 molar ratios) were used for
transfection. For large-scale rAAV preparations, 40×15 cm
plates, each containing 5×106 293 cells were used, and a
single-step gravity-flow column purification method was
carried out, as previously
described[24]. The eluted rAAV was aliquoted and stored at -80 °C. To determine the titer of
the AAV vectors, 5 µL of the rAAV vector was digested with
5 U DNase I at 37 °C for 1 h followed by digestion with 100 µg
proteinase K in proteinase K buffer for 1 h at 37 °C. The
reaction was then extracted with an equal volume of
phenol/chloroform/isoamyl alcohol, and the titer of
rAAV-AT1-AS and rAAV-GFP was determined by dot blot hybridization with
a 32P-labeled probe[23].
Animals Male Sprague-Dawley (SD) rats weighing
250_270 g were obtained from the Tongji Medical College Animal
Center (Wuhan,Hubei Province, China). All of the animals
were housed at room temperature with 12 h light/dark cycles
and allowed free access to normal rat chow and water ad
libitum. The experimental protocols complied with the
National Institutes of Health Guidelines for the Care and Use of
Laboratory Animals and were approved by the Chinese
Academy of Sciences. The animals were randomly allocated to
the groups of rAAV-AT1-AS, rAAV-GFP and Saline.
Gene delivery The animals were anesthetized
intraperitoneally with pentobarbital at a dose of 40 mg/kg body weight.
rAAV-AT1-AS, rAAV-GFP (about
2×1010 virion particles), or an equal volume of saline was then administered into the SD
rats by tail vein injection. After the injection, the rats were
kept warm under an infrared lamp until they recovered. After
2 weeks, the animals of rAAV-AT1-AS and rAAV-GFP groups
were exposed to a high-salt diet (8% NaCl) and the animals in
saline group received normal diet for 12 weeks, and then
cardiac catheterization was performed for the hemodynamic
measurements before the animals were killed under
pentobarbital anesthesia (40 mg/kg body weight). The hearts,
lungs, brains, aorta, and livers were harvested, snap-frozen
in liquid nitrogen, and stored at -80 °C.
Blood pressure measurements The systolic blood
pressure of the rats was measured with a manometer_tachometer
(Rat tail NIBP system, ADI Instruments, Bella Vista, NSW,
Australia) by cuffing the tails of the animals. The rats were
restrained in a plastic cage for approximately 15_30 min to
acclimatize and were minimally warmed before the blood
pressure determination. Five to ten stable readings of systolic
blood pressure were taken weekly for each animal. All of the
measurements were performed between 21:00 and 24:00. The
systolic blood pressure was represented by the mean of these
stable recordings.
Serum and urine collection Urine samples were
collected for 24 h in metabolic cages with 500 µL toluene to
prevent the decay of the urine. The samples were stored at
-80 °C before the urinary microalbumin concentration was
measured.
Whole heart and kidney weight measurements
At 12 weeks after the high-salt diet, the animals were anesthetized
with pentobarbital and the hearts were removed. The whole
heart, double kidney, and body were then weighed. The
ratios of whole heart/body weight and double kidney/body
weight were calculated.
RT-PCR analysis of rat AT1 mRNA
Total RNA was
extracted from fresh rat tissues using TRIZol reagent (Gibco,
Carlsbad, CA, USA), according to the manufacturer's
instructions. RT-PCR for the rat AT1 transcription level was
then performed using the RT-PCR kit (TaKaRa Biotechnology,
Dalian, Liaoning, China), according to the manufacturer's
instructions. PCR primers specific for rat
AT1 were 5'-GGA TGG CGG GAA GTC TAT T-3' for upstream and 5'-TGG GAA
TCT TTG AGA ACT GG-3'for downstream (1151_1651 bp,
which will not amplify transected antisense cDNA and RNA)
and β-actin 5'-TCC TCC CTG GAG AAG AGC TA-3' for
upstream and 5'-TCA GGA GGA GCA ATG ATC TTG-3' for
downstream.
Urinary microalbumin and sodium measurement
Urinary microalbumin was measured by ELISA. The albumin
antibody-coated and blocked microtiter plates (96 wells),
labeled with albumin antibody, albumin standard, and
O-phenylenediamine(OPD) substrates were stored at 4 °C.
The albumin standard (0.1_6.4 µg/mL) and rat urine samples
were added to individual wells in a total volume of 50 µL
dilution buffer. The labeled albumin antibody (50 µL) was
added to the dilution buffer and mixed gently. The plates
were incubated at 37 °C for 90 min, and after incubation, the
plates were washed 4 times with wash solution. The color
reaction was performed by adding 100 µL freshly prepared
OPD substrate solution at 37 °C for 20 min. Then the plates
were read at 490 nm on an Elx 800 ELISA reader (Biotek
Instruments,Winooski, Vermont, USA). Urinary sodium was
measured at the clinical laboratory of Tongji Hospital using
an automatic analyzer.
Hemodynamic variables The hemodynamic variables
were monitored using a catheter tip manometer (AD
Instru-ments, Bella Vista, NSW, Australia) advanced from the right
carotid artery via the aortic arch into the left ventricle (LV) at
the end of the experiment. Global systolic function was
measured as LV peak pressure (LVPSP), LV end-diastolic
pressure (LVEDP), and the maximum_minimum rate of pressure
increase (dp/dtmax). Global LV end-systole was defined as
the point of minimum dp/dt and LV end-diastole as the
beginning of the sharp upslope of the LV
dp/dt tracing.
Analysis of morphology and collagen deposit
The rat kidneys and hearts were preserved in 4%
phosphate-buffered saline-formaldehyde solution and then embedded in
paraffin. Four micrometer-thick sections were cut and stained
with Sirius Red (collagen stained with Sirius Red), using the
method described by Dobrzynski et
al[25]. All of the sections were evaluated by investigators who were blind to the
experi-ment. The percentage of extracellular matrix (ECM)
production was quantified at the Department of Ultrastructural
Pathology, Tongji Hospital with the use of the HAIPS
pathological imagic analysis system (Wuhan, China). To quantify
the ECM, red staining was expressed as a percentage of the
total tissue area[26].
Statistical analysis The statistical significance of the
difference in the systolic blood pressure between rats
receiving rAAV-GFP and rats receiving
rAAV-AT1-AS was determined by ANOVA. In addition, we used unpaired
Student's t-test to assess the differences in urine
microal-bumin content, the ratios of total heart/body weight, and the
results of the ECM quantification between the rAAV-GFP
and rAAV-AT1-AS groups after gene delivery. The results
were expressed as mean±SEM.
Results
Hypotensive effects of rAAV-AT1-AS
The systolic blood pressure of all animals was monitored weekly before and
after exposed to a high-salt diet. The results showed that a
high-salt diet induced a significant elevation in the systolic
blood pressure up to 132.3±12.9 mmHg
(P<0.01. n=8. ANOVA) in the rAAV-GFP-treated rats compared with the
rAAV-AT1-AS and saline solution-treated rats at 1 week. The
systolic blood pressure kept elevating and the maximal blood
pressure difference was up to 27.8 mmHg. In contrast, in the
rAAV-AT1-AS-treated animals and normal controls, the blood
pressure was stable for the duration of the experiment (12
weeks; Figure 1). These results indicated that
rAAV-AT1-AS delivery prevents the development of high blood
pressure in high-salt diet rats long term.
Hemodynamic variables The hemodynamic variables
were monitored using a catheter tip manometer advanced
from the right carotid artery via the aortic arch into the LV.
First, the intra-arterial blood pressure was recorded (126.5±2.95 mmHg in the normal control, 129.2±7.72 mmHg in
rAAV-AT1-AS vs 148.0±7.57 mmHg in rAAV-GFP,
P<0.01, n=5) indicating consistency with tail artery blood pressure monitoring.
The LVPSP, the left LVEDP, and the maximal/minimum ratio
of LVP (±dp/dtmax) were measured and evaluated. The
results showed that significant differences in
hemodynamics between different groups were observed (Figure 2). In
the rAAV-GFP-treated animals, the LVPSP (190.3±6.02
vs 167.5±4.94 mmHg, P<0.05, n=5),
+dp/dtmax (9703.8±131.4
vs 7837±930.3 mmHg/s, P<0.05,
n=5), and
_dp/dtmax (5830.4±
165.4 vs 4366±353.2 mmHg/s, P<0.05,
n=5) were significantly lowered, and LVEDP increased (4.04±0.07
vs 5.01±0.13 mmHg, P<0.05,
n=5) compared with the normal control. In contrast,
rAAV-AT1-AS treatment prevented the deterioration of the
cardiac function index (190.3±6.02, 9703.8±131.4, 5830.4±165.4, and 4.04±0.07, respectively) without significant
differences compared with the normal control, but was
significantly different compared with the rAAV-GFP-treated animals
(P<0.05; Figure 2). These data indicate that
rAAV-AT1-AS treatment significantly attenuates systolic and diastolic
dysfunction in salt-induced, hypertensive rats as high blood
pressure was reduced.
Attenuation of cardiovascular hypertrophy and fibrosis
Myocardial hypertrophy is an important part of heart injury
resulting from hypertension, and serves as an independent
predictor of cardiovascular events in clinical practice. In
this study, we measured the ratio of heart weight/body weight
to analyze the effects of rAAV-AT1-AS treatment on
myocardial hypertrophy and remodeling. As shown in Figure 3, this
ratio was significantly reduced in the
rAAV-AT1-AS-treated rats compared with the rAAV-GFP-treated animals
(2678.1±105.5 vs 2904.2±34.4 mg/kg, n=8,
P<0.05). Sirius Red staining for collagen I-VI, representing myocardial fibrosis and matrix
proliferation, showed that the red-stained area significantly
increased in the rAAV-GFP-treated animals compared with
the rAAV-AT1-AS-treated rats and normal control groups,
suggesting an increased collagen deposit in salt-induced,
hypertensive rats (Figure 4). Meanwhile, the structures were
deranged and the sizes of the cardiac myocytes were not
uniform in the control rats in LV sections. A further
quantitative analysis of the Sirius Red-stained sections was performed
using HAIPS pathological imagic analysis system and was
expressed as the ratio of collagen to the kidney area
(percent-age of ECM). The results demonstrated that
AT1-AS significantly reduced ECM accumulation compared with the
rAAVGFP-treated animals (4.26±3.51 vs 15.58±6.25% ECM,
n=8, P<0.05; Figure 4D).
AT1-AS treatment decreases urinary microalbumin
secretion An increased albumin concentration in the urine is a
marker of kidney injury, as well as an independent predictor
of cardiocerebral vascular disease. In this study, the
micro-albumin level in the urine was measured by ELISA. Initially,
the urine microalbumin levels had no difference between the
3 groups. However, after exposure to a high-salt diet, the
urine microalbumin level in the control animals receiving
rAAV-GFP reached 2.95 ±0.82 ng/mL and 3.05±0.76 ng/mL,
respectively, at weeks 6 and 12), which was significantly
higher than that in rats receiving
rAAV-AT1-AS (2.65 ±0.69 ng/mL and 2.72±0.86 ng/mL at weeks 6 and 12, respectively,
P<0.05) and saline injection (2.56±0.41 ng/mL and 2.52±0.58
ng/mL, at week 6 and 12, respectively, P<0.05, Figure 5). The
results suggest that rAAV-AT1-AS treatment markedly
attenuates the renal damage induced by hypertension from
a high-salt diet.
Protective effects of AT1-AS on kidneys
To evaluate the beneficial effects of
AT1-AS gene delivery in high-salt diet rats, collagen deposit in the kidneys was examined by Sirius
Red staining. In the rAAV-GFP rats, there was more intense
and diverse red staining in glomerules than that found in the
AT1-AS-treated and normal control animals (Figure 6A_6C).
The ECM accumulation was then quantified and was
markedly lower in the group receiving
rAAV-AT1-AS (11.80%±1.22%) than that in the rats receiving GFP vectors (16.22%±0.85%,
P<0.05; Figure 6D). The results further indicate that
AT1-AS treatment significantly attenuates renal damage induced by
hypertension.
Expression of rat AT1-AS The efficient expression of
antisense AT1R mRNA is crucial for blocking
AT1R and therefore in the present study, the expression of the rat
AT1-AS was analyzed using semiquantitative RT-PCR. Total RNA
was prepared from animal hearts, kidneys, and aorta 12 weeks
after exposure to a high-salt diet. RT-PCR was carried out
using primers specific to rat native
AT1R cDNA (base pair 501) rather than transfected antisense cDNA or its mRNA.
As shown in Figure 7, in the salt-induced, hypertensive
animals (rAAV-GFP group), the AT1R mRNA level was
significantly upregulated in the heart, kidney, and aorta compared
with the normal control group. In contrast,
rAAV-AT1-AS treatment markedly downregulated the transcription of
AT1R in rats compared with the control groups, including both
rAAV-GFP-treated and normal control rats. Furthermore, we
reviewed the AT1R protein expression and found that
AT1R was unregulated in the heart, kidney, and liver of the
salt-induced, hypertensive rats;
rAAV-AT1-AS treatment dramatically attenuated the effect to a lower level than the normal
control (Figure 7). The results suggest that the
rAAV-mediated partial AT1R antisense cDNA delivery was not only
transcripted, but also efficiently blocked the transcription of
AT1R mRNA in the main organs, therefore preventing the
development of hypertension and protecting against
hypertension-induced target organ injury.
Discussion
The AAV has many advantages and properties compared
with other vectors, such as retroviruses
or adenovirus, including safety, the ability to infect both the dividing and
non-dividing cells, and the lack of pathogenesis or
inflammatory response. In the present study, therefore, we
constructed the rAAV with antisense to
AT1R. The rAAV vector has been shown to drive an efficient and prolonged transgene
expression, either via persistent episomal
concatamers[27,28] or by genomic
integration[21,22].
It is well known that a high-salt diet is an important
inducer for hypertension[29_31]. Scientists have found that high
salt can induce the overexpression of
AT1R in vascular smooth muscles both
in vivo and in vitro[7,8], and subsequently
enhance the re-absorption of water and salt; that is why a
high-salt diet induces hypertension. Moreover, high salt can
induce the overexpression of AT1 in the kidneys and promotes
fibrosis and hypertrophy of the kidneys and
heart[32-35]. The clinical applications of a new class of antihypertensive drug
AT1 blockades resulted in efficient reduction, organ
protec-tion, and improved long-term
prognosis[36,37], which provides further support for our hypothesis. Recently, Phillips
et al exploited the AAV and retroviral vectors to
mediate AT1-AS-induced hypotension in SHR and 2K1C
rats[11,38_42], which suggests the possibility of the antisense
AT1 gene to treat hypertension through systemic delivery. However, the
hypotensive effect of rAAV-mediated antisense
AT1 on high-salt diet-induced hypertension has not been documented.
In this study, we used rAAV, an efficient and rapid
expression vector, to mediate partial
AT1-AS (660 base pairs) delivery. The results showed that 1 bolus injection of
rAAV-AT1-AS prevented a rise in blood pressure in high-salt
diet-induced hypertensive rats in the long term, and this effect
can come as early as 1 week after injection.
RT-PCR showed that systemic rAAV-AT1-AS delivery
markedly reduced the transcription level of
AT1 in the heart, aorta, and kidney of rats in the long term, which not only led
to a sustained reduction in the systolic blood pressure, but
also significantly decreased the urine levels of microalbumin
and protected the kidneys from injury and fibrosis of
glomerulus induced by hypertensive rats. A morphological
analysis showed that the damage in the rAAV-GFP-treated rats
also included tubular dilatation, loss of brush border in the
proximal tubule, and glomerular sclerosis. Large areas of
intense focal fibrosis in the cortex and the medulla vascular
area, indicated by ECM staining, were observed in the
control group, whereas AT1-AS gene delivery greatly
attenuated all these renal damages and reduced the size and
number of protein casts present in the tubules.
Previous studies have documented that high salt plays
an important role in cardiovascular remodeling. The
mechanisms through which high salt induces cardiovascular and
renal remodeling may be direct biological
effects[33_35]. Pachori et al administered the retroviral vector containing
AT1R antisense in the neonatal rat, which resulted in a
significant attenuation of
cardiac hypertrophy despite its
failure to normalize high blood pressure in renin-transgenic
rats[43]. In our study,
rAAV-AT1-AS treatment significantly
attenuated cardiomyocyte diameter and fibrosis, as observed by
reduced ECM staining, as well as homodynamic variable
deterioration and a marked alleviation of diastolic and
contractile dysfunction compared with the rAAV-GFP-treated
animals. The results show that the long-term, stable
expression of the AT1-AS treatment efficiently protects the heart
and improves cardiac function in salt-induced, hypertensive
rats.
In summary, this study shows that the delivery of
rAAV-mediated partial antisense cDNA to the
AT1R has the potential for
chronically preventing hypertension in high-salt diet
animals. The hypotensive effect was sustained and persisted
for over 3 months in the high-salt diet rats,
and protected the heart and kidney from damage.
These findings suggest the feasibility of
rAAV-AS for the gene therapy of hypertension
and its complications. No adverse effects induced by rAAV
injection were observed during the course of the study. The
high level of expression from rAAV makes it attractive for
human gene therapy. More experiments are needed to study
the immunological aspects and the influence of the
overexpression of the AT1-AS gene on diverse biological
processes before this approach is utilized clinically in the
therapy of hypertension and other cardiovascular and renal
diseases.
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