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Introduction
Tissue kallikrein belongs to the serine proteinase family
that cleaves a low molecular weight kininogen substrate to
produce a vasodilator kinin peptide[1]. The binding of kinin
to the bradykinin (BK) B2 receptor activates secondary
messengers in target tissues and triggers a wide spectrum of
biological effects, such as vasodilation, vasoconstriction,
increase in vascular permeability, and the inhibition or
stimulation of cell growth[2_5]. The vasodilatory action of the
kallikrein-kinin system counterbalances the vasoconstrictive
action of the renin-angiotensin system (RAS). Because of
these functions, human tissue kallikrein (HK) has been
explored for hypertensive gene
therapy[5_7]. Several studies have demonstrated that the muscle delivery of HK gene can
modulate blood pressure and ameliorate the secondary
syndromes associated with hypertension in
rats[8_12].
The recombinant adeno-associated virus (rAAV) vector
is a promising for gene therapy. It can direct long-term
transgene expression in many tissues, including the liver
and muscles, in many animal studies. Since the
adeno-associated virus (AAV) itself has not yet been found to be
associated with any known human diseases, it is presumably safe
for clinical use. In both animal experiments and clinical trials,
rAAV vectors exhibited low vector toxicity. The prolonged
transgene expression via persistent episomal concatamers
also alleviates the fear of random insertion mutagenesis in
the target cells.
Our previous study demonstrated that the systemic
delivery of the rAAV vector-mediated HK gene resulted in
stable, long-term expression and a sequential marked
decrease in systolic blood pressure. It also attenuated organ damage
due to hypertension in spontaneously hypertensive rats
(SHR)[13]. Although intravenous rAAV delivery allowed for
the efficient transduction and secretion of the target gene
product, the systemic delivery of the AAV vector into the
liver may not be an ideal method due to inherited risks. In a
recent clinical trial with hemophilia B, it was found that liver
complications could lead to a disappearance of the transgene
expression. Because HK is a secretary enzyme protein that
is secreted by cells into plasma or tissue fluid and can
function systemically, we explored the possibility of delivering
AAV_HK via intramuscular administration. In this study,
we demonstrated that local rAAV-HK administration can lead
to a long-term, stable expression of the HK protein and a
stable reduction in the blood pressure of SHR , as reported
in our previous study via intravenous
delivery[13]. The results of the present study suggest that
skeletal muscles may be a promising HK-producing organ for gene therapy.
Materials and methods
Plasmid construction The rAAV vector plasmid
pXXUF1, the rAAV plasmid carrying LacZ as a report gene pdxII-LacZ,
adenovirus helper plasmid pXX6, and packaging plasmid
pXX2, were described
previously[14]. The pXXUF1 contains 2 AAV inverse terminal repeats, a cytomegalovirus (CMV)
promoter, and a poly A sequence. The plasmid
pXX6 was constructed by inserting the large
ClaI/SalI fragment of pXX5 into plasmid pBluescript KS
(+). An 860 base pair NotI fragment containing the HK gene open reading frame was
subcloned into pXXUF1 in the downstream of the CMV
promoter to obtain plasmid pUF1-HK, which is used for the
production of the rAAV vector expressing the HK gene.
rAAV vector production, purification, and titer
determination For the rAAV production, human 293 cells were grown
in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand
Island, NY) supplemented with 10% fetal bovine serum (FBS,
Gibco, USA) and antibiotics in a humidified environment
with 5% CO2 at 37 °C. At 1_2 h prior to transfection, the cells
(70%_80% confluent) were transfected with fresh DMEM
containing 10% FBS. A total of 85 µg plasmid DNA/15 cm
dish (molar ratio of pUF1-HK or pdxII-LacZ:pXX2:pXX6 was
1:1:1) was added to CaCl2 solution (0.25 mol/L) and then
gently mixed with 2×BES-buffered saline
(pH 7.0). The
resulting DNA/calcium/phosphate complex was incubated
at room temperature for 30 min before being applied to the
293 cells. At 8_12 h after transfection, the transfection
mixture was replaced with fresh DMEM
containing 10% FBS. The cells were harvested at 48_72 h postinfection. After
low-speed centrifugation on a tabletop centrifuge, the cell
pellets were resuspended in 100 mmol/L NaCl_10 mmol/L
Tris-HCl (pH 8.5) and subjected to freeze-thaw for 4 cycles; the
cell debris was removed by centrifugation.
For a typical large-scale rAAV preparation, approximately
2×108 293 cells (40 15 cm dishes) were used for the transfection. For the purification,
we adopted the single-step gravity-flow column purification
procedure, which was described by Auricchio et
al[15]. The eluted rAAV was aliquoted and stored at -80 ºC for vector
administration.
To determine the titer for the rAAV-HK and rAAV-LacZ
vectors, 5 µL rAAV was digested with 5 U DNase I at 37 ºC
for 1 h. After DNase inactivation, 2×proteinase K buffer and
100 µg proteinase K were added and incubated with rAAV
solution for 1 h at 37 °C, followed by extraction with an equal
volume of phenol/chloroform/isoamyl alcohol. The
resulting solution, which contained vector DNA, was then used
for dot blot hybridization to determine the titers of
rAAV-LacZ and rAAV-HK[16].
Animal handling and vector
administration The 18 SHR were housed at room temperature
with a 12 h light/dark cycle and allowed normal rat chow and tap
water. All of the animal experimental protocols were
approved by the Institutional Animal Research Committee of Tongji
Medical College (Wuhan, China) and were carried out
according to the guidelines of the National Institutes of Health. Two-month-old
male SHR weighing 180_220 g were used for the gene
delivery experiment. rAAV-HK or rAAV-LacZ (about
1×1011 viron particles in 1 mL saline solution) was gently injected into
both sides of the femoral area of the SHR at 3 sites, and the
control animals received an injection of equal saline volume
(n=6 for each group). All the animals were killed 28 weeks
after rAAV delivery under pentobarbital anesthesia (50 mg/kg body weight) and the rats' hearts, lungs, kidneys,
brains, aorta, and livers were collected, frozen in liquid
nitrogen, and then stored at -80 ºC until further analyses. A
portion of the organs was fixed with neutralizing formalin
for the histological analysis.
Systolic blood pressure measurement 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.
This device requires minimal warming of rats (usually 15 min)
before blood pressure determination and a brief period of
restraint in a plastic cage. For every measurement, the
systolic blood pressure was represented as the mean of at least
5 recordings.
Serum and urine collection The 24-h urine samples
were collected in metabolic cages with 500 µL toluene to
prevent the decay of urine. Approximately 1 mL blood was
drawn from the tail vein, and sera were collected after
coagulation and centrifugation. All samples were stored at
-80 ºC before being assayed for kallikrein levels and the
urinary microalbumin concentration.
Echocardiography analysis of heart etiology and cardiac
function of SHR To perform the
echocardiography analysis on the SHR, the animals underwent pentobarbital
anesthesia (50 mg/kg body weight). Echocardiography took place at
the imaging division of Tongji Hospital. The
echocardio-graphy analysis was carried out by 2 echocardiogram
experts independently.
Whole heart and left ventricle weight determination
At 28 weeks' post-HK gene delivery, the animals were killed;
the hearts were collected and weighted. The left ventricle
was weighed after the right ventricular free wall and both
atrias were carefully dissected from the left ventricle. The
inter-ventricular septum was included in the left ventricle for
the weight measurement. The ratios of both whole
heart/body weight and left ventricle/whole heart were then
calculated.
RT-PCR analysis of HK mRNA Total RNA was extracted
from fresh rat tissues using TRIZol reagent (Gibco, Carlsbad,
CA, USA). RT-PCR analysis specific for HK mRNA was
carried out using HK primers (sense:
5'-CCACCATGGGGTTCC-TGGTT-3' and antisense:
5'-CGCGGATCCACATTTGATTT-3'); the β-actin control primers were
5'-GGAGAAGATGACCC-AGATC-3' (sense) and 5'-GATCTTCATGAGGTAGTCAG-3'
(antisense). RT-PCR was carried out following the
instructions of the RT-PCR kit supplied by TaKaRa Biotechnology
(Dalian, Liaoning, China). After first-strand cDNA synthesis,
the PCR reaction was performed in the mixture at a final
volume of 100 µL with 2 µL of the reverse transcription reaction
product in a thermal cycler with a denaturing phase of 1 min
at 94 oC, annealing phase of 40 s at
65 oC, and extension phase of 1 min at 72
oC for 20 cycles.
ELISA analysis of HK concentration The ELISA kit for
HK in animal urine samples was obtained from Dr Chao LEE's
laboratory (Medical University of South Carolina, Charleston,
SC, USA), and ELISA was performed according to the method
described previously[8,13]. To make the biotin-labeled
anti-HK immunoglobulin (IgG), purified rabbit anti-HK IgG was
dialyzed against 0.1 mol/L sodium bicarbonate buffer, pH
9.5, at 4 °C for 24 h and added to 10 mL freshly prepared 0.1
mol/L biotinyl-N-hydroxy-succinimide
ester (dissolved in dimethyl formamide). The reaction was
carried out at room temperature for 1 h and the mixture was
dialyzed against phosphate-buffered saline (PBS). Microtiter plates (96-well) were
coated with non-labeled anti-HK IgG (2
µg/mL, 100 µL/well) overnight at 4°C. The plates
were then blocked with 200 µL PBS (10 mmol/L sodium phosphate, pH 7.4, 150 mmol/L NaCl)
containing 1% bovine serum albumin at 37 °C for 1 h. The
coated plates were washed 3 times with PBS containing
0.1% Tween-20 (washing solution). The purified HK
standard (0.04_2.5 µg) and rat urine or serum samples were
added to individual wells in a total volume of 100
µL PBS containing 0.05% Tween-20 and 0.5% gelatin (dilution
buffer). After incubation at 37 °C for 90 min, the plates
were washed 3 times and 100 µL of 1 µg/mL
biotin-labeled anti-HK IgG was added to each well. After incubation at
37 °C for 1 h, the plates were washed 3 times and 100 µL of
1 µg/mL peroxidase-avidin was applied to each well. The
plates were subsequently incubated at 37
°C for 1 h and washed 5 times
with the washing solution and once with PBS before adding 100 µL freshly-prepared
substrate solution (0.03%
2,2'-azino-bis[3-ethyl
benzthiazoline-6-sulfonic acid]; 0.03%
H2O2 in 0.1 mol/L citrate buffer, pH
4.3). The plates were read at 405 nm on an ELX 800 ELISA
reader (Bio-Tek instruments, Winooski, Vermont, USA).
Analysis of morphology and collagen
deposition A portion of the kidney and heart of each animal were
preserved in 4% PBS-buffered formaldehyde solution and
embedded in paraffin. Four micrometer-thick sections were
cut and stained with hematoxylin-eosin and Sirius Red
(collagen stains red with Sirius Red staining) using the
method described by Dobrzynski et
al[17]. To evaluate and quantify the collagen density in the tissues, sections of the
heart, kidney, and aorta were analyzed by using HAIPS
pathological imagic analysis system after Sirius Red staining, and
the results were expressed as the ratio of collagen to
non-collagen area (percentage of extracellular matrix [ECM]).
Statistical analysis The statistical significance of the
difference in systolic blood pressure among SHR receiving
rAAV-LacZ, rAAV-HK, and saline, respectively, was
determined by ANOVA. In addition, we used unpaired Student's
t-test to assess the differences in the urine microalbumin
levels, HK levels in the urine and serum, the ratios of both
whole heart/body weight and left ventricle/total heart, and
the results of the echocardiography analysis between the
rAAV-LacZ and rAAV-HK groups after gene delivery. The
results were expressed as mean±SEM.
Results
HK expression detection in the muscles post-rAAV-HK
intramuscular vector delivery To assess the expression of
HK post-intramuscular AAV gene delivery, total cellular RNA
was isolated from the heart, renal, lung, liver, and skeletal
muscles at the femoral area at 28 weeks using TRIZol reagents.
RT-PCR was performed using 5 µg total RNA and
oligonucleotide primers designed according to the sequence of the HK
gene. As shown in Figure 1, human kallikrein mRNA was
detected in skeletal muscle tissues at the injection sites in
rAAV-HK-treated rats, but not in other tissues in the HK
group and all the tissues of the rAAV-LacZ-treated rats. A
similar level of β-actin mRNA was detected in the tissues of
both the experimental and control groups. This result
suggests that the muscle tissue is the primary site for HK gene
transcription and protein expression post-intramuscular
rAAV gene delivery.
HK gene delivery leads to the secretion of
immunoreactive HK We next examined the expression of HK in the urine
and blood of the SHR by ELISA. As expected, no
immunoreactive HK was detected in the urine of the control rats
receiving rAAV-LacZ and saline. In contrast, immunoreactive
kallikrein in the urine of rats receiving rAAV-HK vectors
intramuscularly reached 4.86±0.347 ng/mL in 2 weeks and
maintained at a similar level throughout the entire experimental
period (Figure 2A). Correspondingly, HK in the serum of the
testing group receiving rAAV-HK was maintained at a steady
level of 1.12±0.068 ng/mL. It was parallel to the kallikrein
levels in the urine in all the durations of the experiment
although it was lower than that in the urine of the rats
receiving rAAV-HK intravenously[13]. In the serum of the control
animals receiving AAV-LacZ or PBS, only the basal level of
kallikrein was detected, which was likely due to a cross-reaction of antihuman kallikrein antibodies (Figure 2B). The
results demonstrate that rAAV-mediated intramuscular HK
transfer can lead to the persistent and high level secretion of
the HK protein in vivo.
Antihypertensive effect of rAAV-HK in
SHR To study the antihypertensive effect of rAAV-HK delivered
intramuscularly, we monitored the systolic blood pressure
of SHR receiving the control vector and rAAV-HK. In the
control group receiving AAV-LacZ or saline, the systolic
blood pressure of the rats kept rising and reached 180_185
mmHg at week 28 and then stabilized until the end of the
experiments. In contrast, in the rAAV-HK-treated animals,
blood pressure was lowered to 168_178 mmHg. The average
difference between the rats receiving AAV-LacZ and
AAV-HK was 12.6±1.93 mmHg (P<0.05,
n=6, ANOVA). There was no significant difference between the LacZ- and saline-treated
groups (Figure 3). These results indicate that the
intramuscular delivery of rAAV-HK induced a stable and long-term
hypotensive effect and prevented the development of
hypertension in SHR.
Protective effects of HK on kidney histology
To analyze the secondary effects of the intramuscular delivery of
rAAV_HK, we inspected the extent of kidney damage of the
experimental groups by hematoxylin-eosin staining. As shown in
Figure 4, hypertension induced severe damage in the
kidneys of the control SHR, which included tubular dilatation,
atrophy of partial renal tubules, loss of brush border in
proximal tubules and the formation of protein casts, and
glomerular sclerosis and atrophy as observed in other
studies[18,19]. On the contrary, rAAV-HK delivery significantly attenuated
this renal damage induced by hypertension. In the
rAAV-HK-treated rats, the kidney structures, including the renal
cortex and medulla, exhibited little pathological changes
(Figure 4).
Fibrosis of the kidneys was then assessed using Sirius
Red staining. To quantify ECM stained by Sirius Red, the
red staining area was expressed as a percentage of the total
tissue area. This percentage was used as an index of fibrosis.
The results showed that many intense red staining in the
glomerulus and medulla vascular areas in the control SHR,
which represented the collagen deposition in the kidney.
In contrast, the SHR treated with the HK gene showed much
less collagen deposition or ECM accumulation than the
LacZ-treated animals (10.06%±2.94% vs 24.77%±2.08%,
P<0.05, n=6; Figure 5). This analysis suggests that HK attenuated
the collagen deposition or fibrosis in the SHR kidney, which
is the primary mechanism in which rAAV-HK treatment is
able to protect the kidney from hypertensive injury.
Attenuation of cardiovascular remodeling and cardiac
hypertrophy Myocardial hypertrophy is an important part
of heart injury resulting from hypertension as well as an
independent predictor of cardiovascular events in clinical
practice. It is also an important consideration in the therapy
of the hypertension to reverse cardiac
hypertrophy[20]. In this study, we analyzed the effects of rAAV-HK on
hypertensive cardiac hypertrophy. The results are shown in
Figures 6 and 7. First, the ratio of total heart weight to body
weight in the rAAV-HK-treated SHR was significantly lower
than the control SHR (4.378±0.0530 mg/g
vs 5.100±0.0489 mg/g, P<0.05,
n=6). The rAAV-HK delivery also significantly
attenuated the ratio of the left ventricle weight to whole heart
weight compared with the control SHR (0.782±0.0163 g/g
vs 0.834±0.0116 g/g, P<0.05,
n=6; Figure 6). Second, the result of the echocardiography analysis for the rAAV-HK-treated
rats showed the value of the interventricular septum
thickness (IVS; 2.90±0.041 mm in HK rats
vs 3.45±0.050 mm in control rats,
P<0.05, n=6), and the percentage values of
endocardial fractional shortening (FS%; 69.5%±0.646%
vs 62.5%±1.50%, P<0.05,
n=6) were obviously ameliorated compared with those in the control animals (Figure 7). These
results indicated that the heart structure and cardiac
function in the experimental group were significantly more
attenuated than the control groups. Furthermore, a
microscopy observation after Sirius Red staining showed that the
sizes of the cardiac myocytes were not uniform. The
structures were deranged and there was a red-stained area in the
left ventricle section of the heart in the control rats. This
indicated significant fibrosis in the myocardium. rAAV-HK
treatment significantly alleviated cardiac myocyte
hyper-trophy, derangement of structures, matrix prolifera£tion, and
cardiac fibrosis. ECM accumulation expressed as the
percentage of collagen per total cardiac tissue section was less
than that of the control animals (3.40%±2.03%
vs 19.55%±5.20%, P<0.05, n=6; Figure 8). Finally, rAAV-HK treatment
significantly prevented aorta fibrosis. The ECM
accumulation percentage per total aorta tissue section in the HK group
was 19.78%±1.34% compared to 26.26%±1.28% in the
rAAV-LacZ group (P<0.05, n=6).
Discussion
In our previously published study, we documented that
the intravenous administration of rAAV-HK into SHR resulted
in the long-tern expression of the HK gene and a persistent
reduction of blood pressure in rats; it also prevented organ
damage secondary to hypertension, which suggests that
rAAV-mediated HK gene delivery may act as a safe and
effec-tive therapeutic approach in the treatment of hypertension
or other cardiovascular and renal diseases. However, the
concern is whether diverse infection of rAAV in various
organs will induce any unexpected, adverse effects. Thus,
local infection in skeletal muscles is a promising choice. In
the present study, an equal dose of rAAV-HK was
administered intramuscularly to SHR, resulting in a long-term and
stable HK expression. Because tissue kallikrein is a
secretary protein, a single intramuscular injection of AAV-HK, with
same dose as the intravenous injection we previously
administered[13], led to the persistent expression of HK, and
comparable HK levels as shown by RT-PCR and the ELISA
assay for HK. Similar to the results from the intravenous
delivery study, intramuscularly-delivered HK attenuated the
development of hypertension and prevented secondary
damage in the target organs of young SHR. Cardiac hypertrophy,
fibrosis, and dysfunction were reduced. Cardiac remodeling
and dysfunction were confirmed by cardiac hemodynamic
measurements and echocardiography. Moreover, the
kidney was susceptible to hypertension. With
rAAV-HK-mediated gene delivery, renal damage might be
alleviated, which was confirmed by morphological observation and a quantity
analysis of ECM or collagen
deposition[13]. The results demonstrate that
skeletal muscles may be a promising target organ for hypertension gene therapy.
The rAAV vector is one of the most attractive vectors for
gene therapy because of its ability to direct a persistently
high level of transgene expression in both dividing and
non-dividing cells and low vector toxicity. To our
knowledge, no report about the pathogenesis of the rAAV vector has been
published[21]. One of the ideal targets for rAAV gene
delivery is skeletal muscles, which is the largest organ in the
human body, making up of about 40% body weight in adults.
The metabolism of skeletal muscles is very active and its
supplement of blood is abundant. All these features make
this specific organ a promising gene transfection and
transgenic protein-producing
concourse[18,19,22,23]. Previous studies have documented that rAAV vectors allow efficient
gene transfer into the muscles. Recombinant AAV encoding
the dystrophy gene, human α1-antitrypsin, factor IX, and
human erythropoietin gene, all successfully corrected their
related phenotypes in the animal
studies[24,25]. In the current studies, we demonstrated that the rAAV vector is a suitable
vector for delivering another secretary protein HK to reduce
hypertension and tissue organ pathogenesis associated with
hypertension.
Tissue kallikrein is a serine proteinase which can
produce vasodilative peptides, kinin, and
BK[1]. The binding of kinins to the BK B2 receptor activates second messengers in
target tissues and triggers a wide spectrum of biological
effects, such as vasodilation and the inhibition or
stimulation of cell growth[2_4]. The vasodilatory action of the
kallikrein-kinin system counterbalances the vasoconstrictive
action of the RAS[26]. This is the primary reason why HK is
effective in controlling hypertension. Since B2 receptors
express abundantly in the kidney, aorta, and heart
(unpublished data), it is likely that kinin and BK may
directly activate the B2 receptor in these tissues and therefore
attenuate cardiovascular and renal remodeling.
In conclusion, the intramuscular rAAV-HK gene delivery
can result in therapeutic level of HK gene expression
in vivo. The sustained alleviation of hypertension and target organ
protection from hypertensive damage can also be achieved
by a single dose of vector administration. Because the
intramuscular administration of rAAV-HK has the same efficiency
as intravenous rAAV-HK delivery, the skeletal muscles might
be a promising organ for rAAV-HK gene delivery which should
not be ignored. Thus, it warrants further studies to explore
the full potential of rAAV-HK in treating hypertension.
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