© 1997 American Heart Association, Inc.
Articles |
Adenovirus-Mediated Expression of the Secreted Form of Basic Fibroblast Growth Factor (FGF-2) Induces Cellular Proliferation and Angiogenesis In Vivo
From the Molecular Cardiology Unit, Department of Cardiology, Kyushu University School of Medicine, Japan.
Correspondence to Hikaru Ueno, MD, PhD, Department of Cardiology, Kyushu University School of Medicine, Fukuoka 812-82 Japan. E-mail ueno{at}cardiol.med.kyushu-u.ac.jp
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Abstract |
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Abstract Blood supply through collateral arteries is of critical importance in occlusive arterial diseases such as coronary atherosclerosis. Induction of angiogenic growth factor within either the narrowing arteries or jeopardized myocardium may promote angiogenesis in vivo, leading to salvage of ischemic myocardium. We constructed a replication-defective adenovirus (AdCAsFGF-2) coding for human basic fibroblast growth factor (FGF)-2 that is modified, so that its secretion will be facilitated, by tagging a signal sequence derived from FGF-4. A large quantity of FGF-2 was detected in both the cell lysate and culture medium of COS cells infected with AdCAsFGF-2, indicating that FGF-2 was secreted at least partly from the infected cells. The conditioned medium from the infected COS cells stimulated DNA synthesis in and induced cellular proliferation of arterial smooth muscle cells. These effects were eliminated by adenovirus-mediated overexpression of a dominant-negative truncated FGF-receptor type 1. Implantation of a gel of basement membrane proteins containing fibroblasts infected with AdCAsFGF-2 into the ventral subcutaneous space of mice induced extensive cellular proliferation and the formation of functional arterioles. Cells surrounding the vessels were positively immunostained with antibodies recognizing either smooth muscle–specific
-actin or
factor VIII antigen as a marker for
endothelium. These results suggest that
AdCAsFGF-2 may be useful for delivering functional FGF-2 into
tissues and may lead to therapeutic angiogenesis in vivo.
Key Words: adenovirus • gene therapy • angiogenesis • fibroblast growth factor • ischemic myocardium
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Introduction |
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Myocardial infarction is one of the major causes of death in industrial countries.1 2 It may be possible to save the jeopardized myocardium by increasing the blood supply either by dilating the narrowed coronary arteries responsible for the ischemia with a balloon catheter (percutaneous transluminal coronary angioplasty) or by surgically bypassing the narrowed section using an autologous graft of either arterial or venous origin. However, both procedures are effective for relatively large vessels only, and their benefits may be limited by restenosis.2 3 4 5 Another possible method would be to develop a collateral blood supply to take over for the diseased vessels. It is well known that occlusion of coronary arteries leads to the opening or development of collaterals in the human heart.6 Although the mechanisms and the responsible molecules underlying this phenomenon are not fully understood,6 7 angiogenic peptide growth factors such as acidic and basic FGF (FGF-1 and FGF-2, respectively) have been identified in the adult heart8 9 10 11 and in cultured cardiomyocytes.12
FGFs are potent and multifunctional growth factors mainly targeted at cells of mesoneurodermal origin, including arterial SMCs, ECs, fibroblasts, certain epithelial cells, and neural cells.7 13 14 15 FGF is a physiological mesodermal inducer in the early stages of frog development16 and also a physiological dominant growth factor for cardiomyocytes within 1 week of chicken embryogenesis.17 FGF can stimulate the growth of arterial endothelium; increase production of proteases such as plasmin18 and antiproteolysis proteins including plasminogen activator inhibitor-1, partially through activation of latent TGF-ß by plasmin;19 and induce tube formation of ECs.7 Consequently, FGF alone can induce angiogenesis both in vitro and in vivo, and it is thought to be one of the major factors regulating angiogenesis in vivo.7 13 14 Thus, it seems a reasonable assumption that FGF, if efficiently introduced into the ischemic myocardium, may facilitate the effective formation of collaterals and thus induce beneficial effects. In fact, FGF-2 injected into dog coronary arteries20 or given periadventitially in the pig21 reduced the size of an infarction caused by subsequent ligation of the coronary artery. If such a growth factor could be introduced into the diseased area in a site-specific manner as an expression unit of the appropriate cDNA instead of as the protein itself and expressed for a sufficiently long period of time, more efficient and site-specific effects, with fewer systemic side effects, might well be attained.
Recent studies have shown that a recombinant adenoviral vector can achieve remarkably efficient gene transfer both in vitro and in vivo in a variety of tissues and cell types, including arterial wall cells22 23 24 25 26 27 28 29 30 31 32 33 and myocardium.12 34 35 36 37 Using adenoviral vectors, a high level of gene expression can be expected for weeks rather than days. In the present study, we constructed a replication-defective adenovirus expressing human FGF-2 under a potent constitutive promoter (AdCAsFGF-2) and investigated its angiogenic effects both in vitro and in vivo. In contrast to other growth factors, FGF-2 (and FGF-1) does not have conventional signal peptides, and the mechanisms underlying secretion from FGF-2–producing cells are still not fully understood.7 For secretion, some specialized mechanisms might be required. If so, only minimum biological effects of FGF-2 after gene transfer could be seen, since most of the effects of FGF-2 are mediated by binding to and activating of specific receptors on the cell membrane.38 For this reason, in constructing an adenoviral vector (AdCAsFGF-2), we used a modified cDNA of human FGF-2 in which a signal sequence derived from FGF-4 was ligated to the 5' end of the native FGF-2 cDNA. In this way, we hoped to ensure that FGF-2 would be secreted from the infected cells. We have confirmed that COS cells, which usually do not produce endogenous FGF-2, produced and secreted to the medium a biologically active FGF-2 after infection with AdCAsFGF-2. Furthermore, we observed that application of AdCAsFGF-2 induced a marked cellular proliferation and angiogenesis in vivo. This material may well be useful for the efficient delivery of FGF-2 to tissues and may facilitate therapeutic angiogenesis in vivo.
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Methods |
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Preparation of Adenoviral Vector
A recombinant cDNA for the secreted form of human FGF-2 was constructed by the addition of the signal sequence of FGF-4 encoding 22 amino acids to the 5' end of the cDNA of human full-length FGF-2 coding 155 amino acids39 (provided by T. Maciag). Replication-defective E1 and E3 adenoviral vectors were prepared as described previously.33 40 41 42 Briefly, secreted FGF-2 (sFGF-2) cDNA was placed into a cassette cosmid vector under a CA promoter comprising a cytomegalovirus enhancer and chicken ß-actin promoter43 (pAdCAsFGF-2). A recombinant adenovirus was constructed by in vitro homologous recombination in 293 cells using pAdCAsFGF-2 and the adenovirus DNA-terminal protein complex.40 The desired recombinant adenovirus, designated AdCAsFGF-2, was purified by ultracentrifugation through a CsCl2 gradient followed by extensive dialysis. Contamination by the wild-type adenovirus was excluded by the polymerase chain reaction designed for E1 amplification.44 The titer of the virus stock was assessed by a plaque-formation assay using the 293 cells and expressed in plaque formation units. An adenovirus expressing a truncated form of chicken FGF receptor type 145 that holds the entire extracellular domain and the transmembrane portion but only 26 amino acids of the cytoplasmic domain was also constructed (AdCAFGF-TR). This truncated receptor should disable the wild-type FGF receptor functionally as a dominant-negative mutation.16 17 45 The truncated chicken FGF receptor 1 cDNA was tagged with an influenza virus HA epitope (Ser-Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala-Ser-Leu) at its C terminal without making a frame shift and then inserted into the cosmid vector. We also used three control adenoviruses: AdCALacZ, expressing bacterial ß-galactosidase; Ad1w, which did not express any exogenous gene32 33 37 41 42 ; and AdCATGFß-TR, expressing a truncated TGF-ß type II receptor that functions as a dominant-negative TGF-ß receptor.42
Cell Culture
Arterial SMCs and ECs were prepared from the
thoracic aortas of beef cattle by an explant method.33
COS-1 cells, 293 cells, and BALB/3T3 fibroblasts (clone A31) were
obtained from the Japanese Cancer Research Resources Bank. Cells were
cultured in DMEM (GIBCO-BRL) with 10% FBS supplemented with 2
mmol/L l-glutamine, 100 U/mL penicillin G, and 50 µg/mL
streptomycin. SMCs and ECs from passages 3 to 8 were used in this
study. In vitro gene transfer into cells was carried out by incubation
with the adenoviral vector in serum-free medium (DMEM containing 0.05%
BSA, 1 µg/mL insulin, 5 µg/mL transferrin, and
25 mmol/L HEPES [pH 7.4]) for 2 hours at room temperature
under gentle agitation. After washing twice with PBS, cells were
incubated in either growth medium or serum-free medium until
assayed.
Western Blot Analysis
COS cells in 90-mm dishes were infected with AdCAsFGF-2 at
various multiplicity of infection (moi) or left uninfected. Two days
after incubation in the growth medium, the medium was replaced with
serum-free medium (7 mL/90-mm dish), and cells were incubated for a
further 22 hours until lysed in RIPA buffer (50 mmol/L
NaCl, 30 mmol/L sodium pyrophosphate, 50 mmol/L
NaF, 5 mmol/L EDTA, 10 mmol/L Tris, pH 7.4, 1%
Triton X-100, 1 mmol/L PMSF, 0.2 U/mL aprotinin, 10
mmol/L pepstatin A, and 25 mmol/L leupeptin). The
culture medium was also collected. The cell lysates and culture media
were treated with heparin-Sepharose beads (Pharmacia) 4 hours at 4°C
and washed three times with 1.0 mol/L NaCl buffered with
100 mmol/L Tris-HCl (pH 7.4). The precipitates were
subjected to SDS-PAGE (15%) and transferred onto polyvinylidene
difluoride membranes (Millipore). A recombinant human FGF-2
protein (Intergen) was also used as a positive control and molecular
marker (18 kD). The membrane was probed with a mouse monoclonal
antibody against human FGF-2 (Ab 98, a gift from Takeda Research
Laboratory, Oosaka) and then visualized with an alkaline
phosphatase–conjugated anti-mouse IgG and chromogenic
reagents (Promega).
COS cells were infected with AdCAFGF-TR at various moi or left uninfected, and lysed in RIPA buffer after incubation for 3 days. The lysates were analyzed by immunoblotting with either a monoclonal anti-HA epitope antibody (12CA5) or an extracellular FGF receptor antibody45 after partial purification with wheat germ agglutinin-Sepharose (Pharmacia) and separation by SDS-PAGE (8%). The reactive proteins were visualized as described above.
Measurement of DNA Synthesis
The serum-free conditioned medium (7 mL/90-mm dish) was prepared
as described in the previous section from COS cells infected with
either AdCAsFGF-2, AdCALacZ, or Ad1w at various moi. Confluent SMCs,
ECs, or 3T3 fibroblasts in 24-well plates were incubated in serum-free
medium for 50 hours and then challenged for 20 hours with one of the
conditioned media (200 µL per well) plus fresh serum-free medium (200
µL per well). Cells were then pulsed for 4 hours with 1 µCi/mL
[3H]thymidine (DuPont-NEN). The incorporation of
[3H]thymidine into the trichloroacetic acid–insoluble
material was measured using a scintillation counter. Some cells had
been infected with AdCAFGF-TR at various moi 2 days before the
conditioned media were applied.
Cell Proliferation Assay
SMCs were infected with either AdCAsFGF-2 (10 moi), Ad1w (10
moi), AdCAsFGF-2 (10 moi) plus AdCAFGF-TR (100 moi), AdCAsFGF-2 (10
moi) plus Ad1w (100 moi), or AdCAsFGF-2 (10 moi) plus AdCATGFß-TR
(100 moi). The next day, cells were harvested and plated sparsely in
serum-free medium. The number of cells in fixed fields that were
randomly selected (four fields per dish; two dishes for each group) was
counted daily under a microscope.
In Vivo Angiogenesis
BALB/3T3 cells were infected with either AdCAsFGF-2 or AdCALacZ
at moi 30 or left uninfected, and incubated in growth medium. Three
days later, cells (5x106) were harvested using trypsin,
washed twice with growth medium, resuspended in 100 µL of ice-cold
DMEM containing 10% serum, and then mixed with 500 µL of an ice-cold
gel of basement membrane proteins (growth factor–reduced Matrigel,
Becton Dickinson) on ice. BALB/c male mice (5 weeks of age) were
anesthetized by inhalation of diethyl ether, and the ventral
skin was shaved. Infected cells in Matrigel (total volume, 0.6 mL) were
injected into a subcutaneous space. In some experiments, adenoviral
vectors (2x108 plaque formation units) alone were mixed in
Matrigel and injected into mice. Seven days later, the mice were killed
and the gel plugs fixed in formaldehyde. The fixed gel was embedded in
paraffin, sectioned at 5 µm, and stained with hematoxylin-eosin.
Some sections were subjected to immunohistostaining with primary
antibodies recognizing either SMC-specific -actin (from
Boehringer) or von Willibrand factor (factor VIII antigen, from
DAKO), using a biotinylated rabbit anti-mouse IgG antibody (Nitirei) as
a secondary antibody, peroxidase-labeled streptavidin, and
diaminobenzidine. The cells were lightly counterstained with
hematoxylin. Representative sections were photographed
by a technician blinded to treatment regimen. All animals were treated
under protocols approved by Kyushu University animal care
committees.
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Results |
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Expression and Secretion of FGF-2 From Cells Infected With AdCAsFGF-2
We constructed a replication-defective adenovirus (AdCAsFGF-2) expressing a modified form of human FGF-2 that should be secreted efficiently from infected cells. COS cells, which do not normally produce FGF-2 at a detectable level, were infected with AdCAsFGF-2 at various moi and incubated in growth medium for 48 hours and then in serum-free medium for a further 22 hours. The cell lysates and culture media were separately analyzed by immunoblotting with an antibody against human FGF-2 after a partial purification with heparin-Sepharose followed by SDS-PAGE. FGF-2 of 18 kD and of various sizes (18, 20, and 24 kD) was detected in both the lysates and the culture media (Fig 1
). The results showed that the infected
COS cells produced FGF-2 and that at least some FGF-2 was indeed
secreted from the cells into the medium. We hypothesize that different
degrees of glycosylation may result in production of FGF-2 of
various weights in the medium, although we do not know the exact
mechanisms. The morphology of the AdCAsFGF-2–infected cells under
light microscopy was no different from that of cells infected with
AdCALacZ. Moreover, COS cells infected with AdCAsFGF-2 excluded trypan
blue, indicating that the cells were alive.
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Cells Infected With AdCAFGF-TR Become Refractory to FGF-2
We also constructed AdCAFGF-TR expressing a truncated FGF receptor
1 that should function as a dominant-negative FGF
receptor.16 17 45 BALB/3T3 cells were infected with
AdCAFGF-TR at various moi, and cell lysates were analyzed by
immunoblotting with either an antibody recognizing the
HA epitope (Fig 2A) or an antibody
against the extracellular domain of FGF receptor45 (Fig 2B). The truncated FGF receptor was expressed in a moi-dependent
manner.
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BALB/3T3 cells were infected with AdCAFGF-TR at various moi, AdCALacZ
(30 moi), or left uninfected. Two days later, submaximal concentrations
of either FGF-2 (10 ng/mL) or PDGF-BB (30 ng/mL) were
applied and DNA synthesis was measured (Fig 3). DNA synthesis in response to FGF-2
decreased in a moi-dependent manner in cells infected with AdCAFGF-TR.
Cells infected with AdCAFGF-TR at 30 moi became completely unresponsive
to FGF-2, whereas the infected cells remained fully responsive to
PDGF-BB. These data indicate that AdCAFGF-TR directs cells to express a
dominant-negative FGF receptor and that signaling by FGF-2 is
specifically abolished in the infected cells, as previously
reported.45
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Secreted FGF-2 From Cells Infected With AdCAsFGF-2 Is
Biologically Active
To confirm that the secreted FGF-2 was biologically active, the
conditioned medium from COS cells infected with AdCAsFGF-2 was added to
bovine SMCs, and DNA synthesis was measured. As shown in Fig 4, [3H]thymidine
incorporation was enhanced in the presence of the conditioned medium
derived from COS cells infected with AdCAsFGF-2 (3 moi) but not with
AdCALacZ. When cells had been infected with
AdCAFGF-TR, the conditioned medium prepared from
AdCAsFGF-2–infected COS cells failed to elicit DNA synthesis (Fig 4).
This finding is further support that FGF-2 in the supernatant indeed
induced DNA synthesis in cells. Similar effects of the conditioned
medium were also seen in both ECs and BALB/3T3 fibroblasts (data not
shown).
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When SMCs infected with either AdCAsFGF-2 or Ad1w at 10 moi or left
uninfected were plated sparsely in serum-free medium, only SMCs
infected with AdCAsFGF-2 proliferated thereafter (Fig 5). Both uninfected cells and cells
infected with Ad1w exhibited a gradual decrease in cell number. The
proliferative response observed in cells infected with AdCAsFGF-2 (10
moi) was completely eliminated when cells were coinfected with
AdCAFGF-TR (100 moi) (Fig 5). With AdCAsFGF-2 (10 moi) plus
Ad1w (100 moi) or with AdCAsFGF (10 moi) plus AdCATGFß-TR
(100 moi), cellular growth was similar to that seen with
AdCAsFGF-2 (10 moi) alone (Fig 5).
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) or Ad1w (
) at 10 moi, or left uninfected
(control, 
). The next day, cells were plated sparsely in
serum-free medium (day 0). Cell numbers in the fixed fields (four
fields per dish, two dishes for each group) were counted daily under
microscopy. Some cells were coinfected with AdCAsFGF-2 (10 moi) and
either AdCAFGF-TR (AdFTR, 100 moi) expressing a dominant-negative FGF
receptor 1 (
), Ad1w (100 moi 
), or AdCATGFß-TR
(AdTß-TR, 100 moi) expressing a truncated TGF-ß type II
receptor (
). Ratio of cell number to cell number 24 hours after
plating (day 1) is shown as mean±SD (n=8). Another set of experiment
showed a similar result.
These results (Figs 4
and 5) together indicate that biologically active
FGF-2 was indeed secreted from the cells infected with AdCAsFGF-2 and
that AdCAsFGF-2 can stimulate cellular proliferation through autocrine
and/or paracrine loops.
In Vivo Angiogenesis Induced by AdCAsFGF-2
We investigated whether FGF-2 produced by AdCAsFGF-2
could induce in vivo angiogenesis. BALB/3T3 fibroblasts infected with
either AdCAsFGF-2 or AdCALacZ were mixed with growth
factor–reduced Matrigel (a gel of basement membrane proteins) and
injected into the ventral subcutaneous space of BALB/c mice. Uninfected
fibroblasts were used as a control. Seven days later, the gel plugs
were histologically examined. An enormous cell
proliferation and multiple vessel formation, most of which contained
red blood cells inside the lumen, were observed in the gel plug
containing fibroblasts infected with AdCAsFGF-2 (Fig 6E through
6G). Substantially fewer cells and
virtually no vessel formation were found in the gels containing cells
either infected with AdCALacZ or left uninfected (Fig 6A through 6D).
Immunohistostaining with antibodies recognizing either SMC-specific
-actin or factor VIII antigen as a marker for ECs demonstrated that
SMCs and ECs were indeed present around the vessels (Fig 6F and 6G). These results demonstrate that FGF-2 secreted from the
AdCAsFGF-2–infected cells can induce cellular proliferation and
angiogenesis in vivo.
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When Matrigel containing AdCAsFGF-2 alone was injected, vessel
formation was also observed, but only at the marginal area of the gel
adjacent to the surrounding tissues. Neovascularization was not
observed in the center of the gel plug (Fig 7).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In this study, we constructed an adenoviral vector expressing a secreted form of human FGF-2 with the aim of inducing an efficient therapeutic angiogenesis to a specific site as an alternative to the systemic administration of a large quantity of angiogenic growth factor protein. We confirmed that FGF-2 was indeed secreted to the medium from the infected cells (Fig 1
). The secreted FGF-2 induced DNA synthesis in
SMCs (Fig 4). Infected SMCs proliferated in serum-free medium by
autocrine and/or paracrine loops (Fig 5). These effects were abolished
(Figs 4 and 5) in cells that had been infected with an adenovirus
expressing a dominant-negative truncated FGF receptor 1 (Figs 2 and 3),
further supporting the possibility that the increased DNA synthesis and
cellular proliferation were due to FGF-2 secreted from the
AdCAsFGF-2–infected cells. Subcutaneous injection into mice with a gel
containing fibroblasts infected with AdCAsFGF-2 resulted in extensive
cellular proliferation and neovascularization (Fig 6). These vessels
contained red blood cells and were surrounded with SMCs and ECs (Fig 6), indicating that mature arterioles had been formed in vivo. These
SMCs and ECs probably migrated from neighboring tissues attracted to
FGF-2 produced by fibroblasts in the gel plug. However, the possibility
may remain that some of the fibroblasts become differentiated to ECs
and/or SMCs, since fibroblasts have been documented to be progenitors
of ECs in vivo.46
We confirmed that biologically active FGF-2 was indeed secreted to the
medium from AdCAsFGF-2–infected COS cells, which normally do not
produce endogenous FGF-2 (Figs 1 and 4). The amount of
FGF-2 in the medium may be somewhat underestimated, because some
fraction of FGF-2 may be tightly bound with extracellular matrices
rather than floating in the medium. In our study, we could not evaluate
precisely how effective the modification of tagging a signal sequence
on a native FGF-2 would be. There has been a report comparing the
biological properties of adenoviral vectors that express either a
secreted form of FGF-1 (acidic FGF) or a native FGF-1,47
which is the closest member to FGF-2 among the FGF
family.13 14 In that study,47 the FGF-1 was
modified in the same way as FGF-2 was in the present study. The
modified FGF-1 was secreted to the medium to a much greater extent than
the native FGF-1 and was more effective than native FGF-1 in the
induction of both EC proliferation and the formation of a
capillary-like network in vitro, although not much difference was
observed in in vivo angiogenesis.47 Considering that the
two forms of FGF were modified in the same way in that47
and the present study, the modification of the signal sequence on
FGF-2 would be expected to induce similar effects.
The effectiveness of angiogenic growth factors such as FGFs and another potent angiogenic cytokine, VEGF, has been reported in ischemic limb48 49 50 51 and ischemic myocardium20 21 52 53 54 models. For example, Lazarous et al54 investigated the effects of a long-term systemic administration of a large quantity of FGF-2 protein (1.74 mg/d for 4 weeks) on collateral development in a relatively large mammal, the dog, and found that the FGF-2 treatment induced a marked improvement in collateral flow. Furthermore, it has been reported that FGF-155 and VEGF56 stimulated endothelial proliferation, leading to a relining of injured arteries with regenerated endothelium, and also reduced intimal thickening in rat carotid arteries.56 Periadventitial administration of FGF-2 in a chronic ischemic region of the porcine coronary artery restored the ability of the endothelium to induce relaxation in response to various vasoactive substances.57 However, in most studies, large quantities of growth factor proteins were administered daily: This protocol could impose both financial and practical burdens if such a method were to be used therapeutically. In contrast, adenovirus-mediated gene transfer performed only once may be able to produce a sufficient quantity of protein for an extended period of time in a relatively site-specific manner. Gene expression by an adenoviral vector is limited to 2 to 3 weeks in the myocardium34 35 36 37 and 4 to 6 weeks in arterial walls;33 however, this may not be a serious problem, since FGF-2 treatment for 1 week during the period of maximal ischemia was sufficient to induce a major effect on collateral flow.54 This limitation in gene expression may even be advantageous in avoiding unnecessary angiogenesis and minimizing systemic side effects.
Before the clinical value of an adenoviral vector can be fully
assessed, a direct cytopathic effect and inflammatory response
potentially associated with the current E1 and E3 adenoviral vector
should be tested in vivo using large mammals. The potential systemic
side effects observed with a large quantity of FGF protein should also
be tested in the cases of adenoviral vectors in large mammals. These
undesirable side effects include (1) hypotension due to
vasodilation,54 57 58 as well as anemia and a decrease in
circulating platelets54 ; (2) a potential shift of
phenotype of the myocardium from an adult
differentiated type to a fetal dedifferentiated type, as has been shown
in cultured neonatal rat cardiomyocytes59 ; and
(3) fibroproliferative changes in tissues and/or intimal hyperplasia in
injured arteries.60 61 62 63 These side effects are also implied
by the enormous cellular proliferation seen in the present study
(Fig 6). Finally, it will be important to exclude the possibility of
tumorigenesis, since NIH-3T3 fibroblasts transfected to express
constitutively a secreted form of FGF-1 became
tumorigenic.39 In terms of systemic side effects, it would
be interesting to examine whether use of a native FGF-2 might reduce
the risk.
A potential use has been reported for adenoviral vectors that express either FGF-1 (both a native form and a secreted form)47 or VEGF165.64 In terms of their properties, FGF-1 and FGF-2 are mitogenic for not only ECs but also for SMCs and fibroblasts, whereas VEGF acts more like an endothelium-specific growth factor. VEGF has signal peptides for secretion. At present, it is not known which angiogenic growth factor might be most suited for therapeutic angiogenesis. Moreover, it has not been established which, if any, combination might achieve more beneficial effects, although synergistic effect between FGF-2 and VEGF on endothelial proliferation has been reported both in vitro65 and in vivo.66 Furthermore, a combination of proliferative growth factors, such as FGF and VEGF, and a growth-inhibitory factor, TGF-ß, may be interesting, since TGF-ß potentiated the effect of FGF-2–and VEGF-induced angiogenesis in vitro, depending on its concentration.67 68 On the other hand, the potential fibrosis in response to FGF60 61 62 63 might be attenuated by a coadministration with an adenoviral vector expressing a dominant-negative TGF-ß receptor that can abolish the diverse signaling by TGF-ß, including the transcriptional activation of extracellular matrix proteins.42 Some of these interesting issues are under investigation in our laboratory.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan and by grants from Sandoz Foundation for Gerontological Research (Switzerland), Mochida Memorial Foundation for Medical Science (Tokyo, Japan), and Takeda Research Foundation for Metabolic Diseases (Oosaka, Japan) to H. Ueno. We thank Dr T. Maciag (American Red Cross Hospital), Dr I. Saito (University of Tokyo), and Takeda Research Laboratory for cDNA, a cosmid vector, and an antibody against FGF-2, respectively. We acknowledge the excellent technical support of S. Nishio.
Received May 5, 1996; accepted January 2, 1997.
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