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Introduction
The treatment of patients suffering from peripheral
arterial occlusive disease remains a considerable clinical
challenge despite advances in both surgical and percutaneous
revascularization techniques. Many patients cannot benefit
from these therapies because of the anatomic extent and the
distribution of arterial occlusion. In such patients, new
therapeutic strategies have been sought to prevent the
development of disabling symptoms related to ischemia, such as
claudication, resting pain, and loss of tissue integrity in the
distal limbs. Therapeutic neovascularization via the
processes of angiogenesis (capillary growth) and arteriogenesis
(collateral artery growth) is a promising new approach for
the treatment of this ischemic
disease[1,2].
Among all angiogenic growth factors, the vascular
endothelial growth factor (VEGF) is one of the well-studied
factors for therapeutic angiogenesis[3]. VEGF exerts its
effect by stimulating the proliferation and migration of
endothelial cells. However, the development of blood vessels in
adult tissues is a complex process in which different growth
factors and cytokines act in concert. VEGF-induced vessels
are often leaky and do not properly connect to the existing
vasculature[4]. Angiopoietin-1 (Ang1), which is responsible
for blood vessel maturation, is another promising candidate
for therapeutic angiogenesis[5]. It recruits pericyte and
smooth muscle cells to stabilize and mature newly-formed
blood vessels[6]. Combination gene therapy with several
growth factors is a rational approach to creating more stable
vessels for functional improvement in ischemic tissues. The
combination of VEGF with Ang1 might thus be a more
efficient strategy to develop functional and mature vasculature
in the treatment of ischemic
diseases[7]. The co-administration of plasmid VEGF and plasmid Ang1 led to enhanced
arteriogenesis in the ischemic
myocardium[8].
For therapeutic angiogenesis, several different strategies
have been examined. In some cases, the recombinant
protein was tested. In others, gene transfer using naked DNA
or adenoviral vectors was performed, but the recombinant
protein is limited by the relatively short circulating half-life,
as well as by the large quantity needed for therapeutic
effects[2]. The transduction efficiency of naked DNA is
significantly lower compared with other
methods[9], and adenoviral vectors lack sustained expression and antigenicity
against viral proteins. Adeno-associated virus (AAV)
vectors have a number of attractive features, including
non-pathogenicity and the ability to transduce skeletal muscles
efficiently, resulting in prolonged gene
expression[10,11]. Although most people are seropositive for the virus, the
immune response is limited to the secretion of non-neutralizing
antibodies[12].
For the combination of VEGF and Ang1 in the treatment
of ischemic diseases, we constructed the AAV vectors
simultaneously encoding human VEGF165 and Ang1
(AAV-VEGF/Ang1), and performed intramuscular injection of
AAV-VEGF/Ang1 to investigate their therapeutic effect in a rabbit
hind-limb ischemic model.
Materials and methods
Plasmid construction and vector production The
construction of pAAV-VEGF, pAAV-Ang1, and
pAAV-VEGF/Ang1 were as previously
described[13]. The structures of these plasmids are shown in Figure 1A. Plasmid
pAAV-VEGF/Ang1 simultaneously encoded 2 growth factors: human
VEGF165 and Ang1, but each of them had its own
independent cytomegalovirus (CMV) promoter and human growth
hormone polyadenylation signal (hGH polyA).
A large quantity of AAV-2 vectors were prepared by
using the AAV Helper-Free System (Stratagene, La Jolla, CA,
USA). Briefly, the AAV vector plasmid was co-transfected
with the pAAV-helper and pAAV-RC into HEK 293 cells by
the calcium phosphated method. Three days later, cultures
including transfected HEK 293 cells and medium were
collected. Ten percent (v/v) chloroform was added to the
collected cultures, and vigorously shaken for 1
h[14]. Then solid NaCl was added until the final concentration was 1
mol/L by shaking at room temperature. The supernatant was
harvested by centrifugation at 12
000×g for 15 min at 4 °C. An appropriate amount of solid PEG8000 was added to the
supernatant to obtain a final concentration of 8%
(w/v) by intermittent shaking at room temperature. The supernatant
was cooled in ice water for 1 h. The precipitated AAV
particles were recovered by centrifugation at 11
000×g for 15 min at 4 °C. The pellets were resuspended in
PBS2+ buffer. DNase I (Takara Bio Inc, Otsu, Shiga, Japan) and RNase
(Sino-American Bio Co, Beijing, China) were added to the culture
and incubated for 30 min. An equal volume of chloroform
was added to the suspension which was then shaken
vigorously for 2 min. The organic and aqueous phases were
separated by centrifugation at 12
000×g for 5 min at 4 °C. The aqueous phases containing the AAV vectors were collected
and filtered through 0.2 µm filters (Sartorius AG, Goettingen,
Germany). Disposable hollow-fiber tangential-flow filtration
devices (8 in, 100 kDa, Amersham Biosciences, Piscataway,
NJ, USA) were used to concentrate and diafilter AAV
vectors with the buffer (100 mmol/L sodium citrate, 10 mmol/L
Tris, pH 8.0, 315 mOsm)[15]. Thus, we obtained concentrated
and purified AAV-VEGF, AAV-Ang1 and AAV-VEGF/Ang1.
The vector particle titer was determined by quantitative DNA
dot-blot hybridization of the DNase I-treated vectors.
Rabbit hind-limb ischemia model and gene
transfer Male New Zealand rabbits (weighing 2.0_3.0 kg) were
obtained from the Experimental Animal Center of Wuhan
University, China and were housed under standard
conditions (temperature: 21±1 °C; humidity: 55%_60%) with food
and water continuously available. The care of rabbits
complied with the Guide for the Care and Use of Laboratory
Animals. All animal protocols followed the
recommendations and guidelines of the National Institutes of Health and
were approved by the Wuhan University Animal Care and
Use Committee.
The rabbit hind-limb ischemia model was constructed as
previously described[16,17]. The rabbits were
pre-anesthetized with ketamine (50 mg/kg) and xylazine (5 mg/kg),
incubated, and mechanically ventilated with a mixture
of 1.5% isoflurane and oxygen. The femoral artery was then
excised from its proximal origin as a branch of the external
iliac artery to the point distally where it bifurcated into the
saphenous and popliteal arteries.
Ten days after surgery, the rabbits were randomized to
receive an intramuscular injection of AAV_VEGF
(n=8), AAV-Ang1 (n=8), AAV-VEGF/Ang1
(n=8) or PBS (n=8) with a 25 gauge needle into 5 different sites
(0.5×1013μg/250 μL, 250 μL/site) in the major thigh muscle of the ischemic hind-limbs.
Blood flow measurement Blood flow in the popliteal
artery of ischemic and normal hind-limbs was measured at rest
with an Aspen Advanced Doppler ultrasound device (Acuson, Siemens Medical Solutions, Mountain View, CA,
USA), using a small L10 transducer 8 weeks after the
injec-tion, and was valued as a ratio of the contralateral limbs.
Three separate measurements were done for each rabbit at
every time point, and the results were averaged.
RT-PCR analysis The rabbits were sacrificed 8 weeks
after the injection. The total cellular RNA of the muscle
tissues of the injected limbs and remote tissues (brain, heart,
liver, spleen, kidney, and testes) was isolated using Trizol
Reagent (GIBCO, Gaithersburg, MD, USA). Extracted RNA
was treated with DNase I (Takara Shuzo, Tokyo, Japan) to
eliminate DNA contamination. First-strand cDNA was
synthesized by random hexamer (Invitrogen Corp, Carlsbad, CA,
USA). The PCR amplifications were performed using human
VEGF165 and Ang1 specific forward primers, and specific
reverse primer for hGH polyA. GAPDH was detected by
RT-PCR as an internal control. All primers were described as
follows: human VEGF165 specific forward primer
5'-GACCCT-GGTGGACATCTTC-3'; human Ang1 specific forward primer
5'-TAACAGGAGGATGGTGGTTTGATGCTTG-3'; hGH polyA specific reverse primer
5'-ATGCCTGGAATCCCAA-CAACT-3'; GAPDH primers: forward
5'-TCACCATCTTCC-AGGAGCGA-3'; reverse 5'-CACAATGCCGAAGTGGTCGT-3'.
Western blot analysis Eight weeks after the injection,
muscle samples from the thigh muscle of the ischemic limbs
were harvested and resuspended in lysis buffer (1% Nonidit
P-40, 50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 200 U/mL
aprotinin, 1 mmol/L phenylmethanesulfonyl fluoride). The
tissue lysates (50 mg of protein) were separated by 12%
polyacrylamide gel electrophoresis and blotted onto
poly-vinylidene difluoride membranes. Immunoblotting was
performed with antibodies against human
VEGF165 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA) or human
Ang1(Sigma-Aldrich Corp, St Louis, MO, USA).
Histological assessment Eight weeks after the injection,
tissue sections from the thigh muscle of ischemic limbs were
harvested. Muscle samples were embedded in optimal
cutting temperature (OCT) compound (Sakura Finetek USA, Inc,
Torrance, CA, USA), snap-frozen in liquid nitrogen, and cut
into 5 μm thick sections. To detect the expression of VEGF
and Ang1, some tissue sections were immunostained with
antibodies against human VEGF165 or Ang1.
Some sections were stained with hematoxylin. To detect
capillary endothelial cells, tissue sections were stained for
alkaline phosphatase with an indoxyltetrazolium method as
previously described[18]. Capillaries stained dark brown were
scored as positive. A total of 20 different fields were
randomly selected, and the number of capillaries was counted
(mean number of capillaries per square millimeter).
To identify arterioles and differentiate them from
capillaries and veins, tissue sections were immunostained for smooth
muscle α-actin (α-SMA). The monoclonal antibody against
α-SMA (DAKO, Kyoto, Japan) was applied at a 1:500
dilution after blocking with 1% normal horse serum.
Subsequent incubation with biotinylated horse anti-mouse IgG
(Vector Laboratories, Burlingame, CA, USA) and an ABC
Elite kit (Vector Laboratories, USA) was performed. The
number of α-SMA-positive vessels was counted in a similar
way to that for capillary density.
Serum levels of human VEGF165 and
Ang1 To assess the systemic levels of human
VEGF165 and Ang1, blood samples were carried out 8 weeks after the injection. Serum
concentrations of VEGF165 and Ang1 were measured by ELISA
using a VEGF165 ELISA kit and an Ang1 ELISA kit (R&D
Systems, Minneapolis, MN, USA).
Evans Blue permeability assay Eight weeks after the
injection, a modified Miles permeability assay was performed
as described[19]. The rabbits were anesthetized and Evans
Blue-PBS (30 mg/kg) was injected (iv). The rabbits were
killed 30 min after the injection of Evans Blue and were
perfusion-fixed with 1.5 L of 1% paraformaldehyde in 0.05 mol/L
citrate buffer (pH 3.5) via the left ventricle. After the rabbits
were killed, the muscles were dissected, washed, weighed,
and extracted in formamide at 55 °C overnight. The Evans
Blue absorbance of the formamide was calculated in a
spectrophotometer set at 610 nm and the ratio between
transduced and intact muscle samples was calculated.
Statistical analysis Data were expressed as mean±SEM.
Statistical comparisons were performed using ANOVA
followed by Fisher's test. Values of P<0.05 were considered to
be statistically significant.
Results
Expression of VEGF and Ang1 To confirm human
VEGF165 and Ang1 gene expression in transduced rabbit ischemic limb
muscles, RT-PCR, Western blot, and immunohistochemical
analysis were performed 8 weeks after injection. As shown
in Figure 1, the size of the PCR products for
VEGF165 in groups AAV-VEGF and AAV-VEGF/Ang1, Ang1 in groups AAV-Ang1
and AAV_VEGF/Ang1, and GAPDH were 852, 788, 621, 504,
and 293 bp, respectively. The molecular weights of human
VEGF165 and Ang1 were 42 and 70 kDa, respectively. Skeletal
muscle cells positive for human VEGF or human Ang1 was
stained brown, as shown in Figure 2. All these data
demonstrated that human VEGF165 was expressed in groups
AAV-VEGF and AAV-VEGF/Ang1, but not in groups AAV-Ang1
and PBS, and that human Ang1 was expressed in groups
AAV-Ang1 and AAV-VEGF/Ang1, but not in groups AAV-VEGF and PBS.
Blood samples were also obtained from the peripheral
veins of rabbits 8 weeks after injection and analyzed with the
VEGF ELISA kit specific for human
VEGF165 and the Ang1 ELISA kit specific for human Ang1. Non-human
VEGF165 and Ang1 were detected in plasma, and no human
VEGF165 and Ang1 mRNA could be detected by RT-PCR from remote
organs (brain, heart, liver, spleen, kidney, and testes) in the
AAV-treated rabbits 8 weeks after injection. All these
findings demonstrated that 2 proteins could be simultaneously
encoded in 1 AAV vector and co-expressed in transduced
tissues without ectopic expression.
Improvement of blood flow Eight weeks after the injection,
we assessed the blood flow in the popliteal artery of ischemic
and normal limbs with the Doppler ultrasound device. As
shown in Figure 3B, the ratio of mean ischemic/normal blood
flow in group AAV-VEGF/Ang1 (0.78±0.04) was the highest
compared with groups AAV-VEGF (0.67±0.04),
AAV-Ang1 (0.49±0.04), and PBS (0.47±0.04). Intramuscular
administration of AAV-VEGF/Ang1 could obviously improve the blood
flow of ischemic hind-limbs.
Promotion of neovascularization The muscle samples of
rabbit ischemic hind-limbs were examined histologically 8
weeks after the gene transfer. When histological sections
were stained with hematoxylin, we found that the muscles of
group AAV-VEGF or AAV-VEGF/Ang1 showed remarkably increased cellularity as compared to that of group PBS or
AAV-Ang1 (Figure 3A). To detect capillary endothelial cells,
tissue sections were stained for alkaline phosphatase. As
shown in Figure 4B, capillary density in transduced muscles
of group AAV-VEGF (1032±80/mm2) or AAV-VEGF/Ang1
(1054±76/mm2) was significantly higher than that of group
AAV-Ang1 (336±26/mm2) or PBS
(340±29/mm2). To identify arterioles positive for
α-SMA, we performed immunohistochemistry with an antibody against
α-SMA. The α-SMA-positive vessel density was significantly increased when
VEGF was co-expressed together with Ang1 in group
AAV-VEGF/Ang1 (Figure 5B). At the same time, we never
observed the formation of angioma-like structures in group
AAV-VEGF or AAV-VEGF/Ang1. Intramuscular
administration of AAV-VEGF/Ang1 could significantly enhance
neova-scularization of ischemic tissue.
Prevention of capillary leakage To test the function of
the observed increase in capillary density, we measured
vascular leakage with Evans Blue dye as previously described.
As shown in Figure 5C, the vascular permeability in group
AAV-VEGF (1.46±0.13) was significantly higher than that in
group PBS (0.15±0.04) or AAV-Ang1 (0.17±0.03) induced by
VEGF, but the permeability was obviously reduced when
VEGF was co-expressed together with Ang1 in group
AAV-VEGF/Ang1 (0.43±0.11). Intramuscular administration of
AAV-VEGF/Ang1 could obviously prevent capillary leakage.
Discussion
For combination gene therapy with VEGF and Ang1 in
the treatment of ischemic diseases, we constructed the
AAV_VEGF/Ang1 viral vectors simultaneously encoding 2
angiogenic growth factors, VEGF and Ang1. These 2 genes had
their own independent CMV promoter and hGH polyA. To
prove the feasibility of this approach, we performed an
intramuscular injection of AAV_VEGF/Ang1. Eight weeks after
the injection, we could detect the co-expression of VEGF
and Ang1 in transduced muscles with RT-PCR and Western
blotting methods, and we also found their synergistic
effects of forming mature vasculature. This finding
demonstrated that 2 proteins could be simultaneously encoded in
one AAV vector and co-expressed in transduced tissues.
This finding had an important application value for other
combination gene therapies.
The most striking change we observed was the increase
in the number of blood vessels present in the AAV-VEGF
and AAV-VEGF/Ang1 injected areas. This event was
somehow expected as far as capillaries were concerned, given the
very well-known properties of VEGF on endothelial cell
proliferation and migration[20] and according to studies that
analyzed capillary formation after intramuscular injection of
adenoviral vectors expressing
VEGF[21,22] or of skeletal myoblasts engineered to secrete this
factor[23,24]. However, the vessels in the AAV-VEGF injected areas were leaky. When
VEGF co-expressed with Ang1 in group AAV-VEGF/Ang1,
the permeability of vessels was remarkably reduced.
Consistent with that VEGF is a strong inducer of vascular
permeability[25], Ang1 has been shown to be essential for the
maturation of blood vessels during embryonic
development[26] and for the mitigation of vascular leakage promoted by
inflammation and VEGF in the adult
vasculature[27].
In the present study, we also found that the α-SMA
positive vessel density was significantly increased when VEGF
was co-expressed together with Ang1 in group
AAV-VEGF/Ang1. So this demonstrated that VEGF co-expressed with
Ang1 could enhance arteriogenesis processes in ischemic
tissues. Ang1 could recruit and stimulate the formation of
pericyte and smooth muscle cells leading to maturation of
blood vessels. However, in addition to its property of being
a strong endothelial cell mitogen via its Flk-1 receptor, VEGF
also seemed to stimulate smooth muscle cell generation or
migration[28,29]. Consistent with that, co-administration of
plasmid VEGF and plasmid Ang1 led to enhanced
arterio-genesis in the ischemic
myocardium[8].
Another interesting finding of our study was the
observation that muscles injected with AAV-VEGF or
AAV-VEGF/Ang1 showed remarkably increased cellularity as compared
to PBS or the AAV-Ang1 injected groups. The same
findings were also reported by Arsic et
al[30] who found that these cells were positive for CD31 and VEGFR-2, markers of
hematopoietic/endothelial cell precursors, and also positive
for the c-kit/CD117 marker, an antigen that commonly
defines pluripotent bone marrow stem cells. Different
investigators have recently reported that c-kit-positive bone
marrow cells are capable of infiltrating the infracted myocardium
(in which expression of VEGF is probably high) where they
underwent alternate differentiation routes, including
cardiomyocytes, endothelial, and smooth muscle
cells[31_33]. In addition, circulating endothelial progenitor cells of bone
marrow origin that had been isolated were incorporated in
sites of myocardial
neovascularization[34]. Altogether, these
observations suggest that circulating stem cells might sense
ischemic, damaged tissue, or VEGF and migrate to these
areas where they promote tissue formation by a mechanism
that is probably different from angiogenic sprouting and
might resemble embryonic vasculogenesis.
A potential problem of our study was that the prolonged
and high-level expression of VEGF may result in the
formation of hemangioma. Hemangioma has been seen in the heart
injected with plasmid[35] and retroviral vector-mediated VEGF
gene transfer[24,36]. Retroviruses could mediate transgene
expression at high levels in transduced
tissue[24], and high concentrations of VEGF in local areas could induce
exaggerated angiogenesis and angioma formation. The injection of
a large dose of VEGF plasmid (500 mg of plasmid DNA) to
the ischemic rat heart induced angioma formation. No
angioma was seen in a rat model when a smaller dose (125 mg
of plasmid DNA) was used[35].
Springer et al[24] reported that high levels of serum VEGF (200 mg/mL) caused
hemangiomas in adult skeletal muscles, and that low serum levels
(30 mg/mL) did not cause vascular malformations, but were
sufficient to induce angiogenesis in ischemic muscles.
Hence, it was important to control the level of VEGF
expres-sion. In the present study, AAV-VEGF/Ang1 induced
angiogenesis in the local ischemic environment, but no angioma
was observed and no hVEGF165 was detected in rabbit serum.
These differences were most likely related to AAV gene
transfer biology, in which the onset of growth factor expression
was progressive and the peak levels of the produced factor
were lower than those of other vector
systems[11]. This suggested that AAV-mediated VEGF expression was not as high
as those mediated by adenoviral and retroviral vectors, and
yet it was enough to induce new vascular formation in the
ischemic myocardium. Previous studies have shown that
transduction with AAV vectors could result in gene
expression lasting >1 year[37]. Although the dose of
AAV-VEGF/Ang1 used in this experiment did not induce angioma
formation, it was possible that long-term expression of VEGF
could eventually lead to angioma formation. A method of
circumventing this possibility was to add the hypoxia
response elements found in erythropoietin or VEGF genes
to control the expression of VEGF which could be activated
under hypoxia and turned off once hypoxic conditions had
been resolved[38,39]. Thus, AAV vectors combined with an
inducible promoter could provide a safe delivery system for
AAV-VEGF/Ang1 in clinical use.
In summary, AAV vectors can simultaneously encode 2
proteins which can be efficiently and stably co-expressed in
transduced tissues without ectopic expression.
AAV-mediated VEGF and Ang1 genes transfer enhances angiogenesis
and arteriogenesis, prevents capillary leakage, and improves
blood flow in a rabbit hind-limb ischemic model.
Intramuscular administration of AAV-VEGF/Ang1 may be a useful
stra-tegy for the treatment of ischemic diseases.
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