© 1995 American Heart Association, Inc.
Articles |
Expression of Vascular Endothelial Growth Factor From a Defective Herpes Simplex Virus Type 1 Amplicon Vector Induces Angiogenesis in Mice
From the Department of Medicine (E.A.M., H.J.F., M.B.), Division of Endocrinology, Diabetes Research Center, and the Departments of Neuroscience (H.J.F.), Albert Einstein College of Medicine, Bronx, NY.
Correspondence to Dr Michael Brownlee, Diabetes Research Center, Albert Einstein College of Medicine, Morris Park Ave, F-531, Bronx, NY 10461.
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Abstract Vascular endothelial growth factor (VEGF) is a secreted endothelial cell–specific angiogenic growth factor. VEGF gene transfer strategies to stimulate focal angiogenesis could be used to ameliorate myocardial ischemia. To induce angiogenesis in vivo, we have constructed a replication-defective herpes simplex virus type 1 (HSV-1) amplicon vector that places the human VEGF-165 cDNA under the transcriptional control of the HSV immediate-early 4/5 promoter (HSVhvegf). Transduction of NIH 3T3 fibroblasts with HSVhvegf resulted in the secretion of high levels of biologically active VEGF, as assayed by microvascular endothelial mitogenesis. By use of an ex vivo protocol, BLK-CL4 fibroblasts were transduced with HSVhvegf or control HSVlac virus (expressing Escherichia coli ß-galactosidase), resuspended in basement membrane extract (matrigel), and coinjected subcutaneously into syngeneic C57BL/6 mice. One week later, the matrigel plugs with HSVhvegf showed a strong angiogenic response, in contrast to the plugs with HSVlac-transduced fibroblasts. These data indicate that transduction with HSVhvegf virus can induce an angiogenic response in vivo and suggest that this is a viable gene therapy approach for tissue ischemia.
Key Words: lymphokines • DNA virus • gene therapy • endothelium • neovascularization
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Introduction |
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Angiogenesis is a complex process by which an organism creates new capillaries.1 Angiogenesis is induced during vascular development, in the corpus luteum, and also under pathological circumstances, such as in solid tumors and after injury.1 As a natural adaptative response to myocardial ischemia, the coronary collateral circulation increases by capillary proliferation.2 However, in most cases this compensatory adaptive process is incomplete.2 A novel approach to ameliorating tissue ischemia is by the local administration of angiogenic growth factors. Such angiogenic therapy has been shown to increase collateral circulation,3 4 limit myocardial loss in a canine infarct model,4 and increase revascularization in a rabbit ischemic hind limb model.5 6
Several growth factors have been demonstrated to be angiogenic,1 7 the best characterized being the fibroblast growth factor (FGF) family7 8 and the vascular endothelial growth factor (VEGF) family.9 10 11 12 VEGFs are endothelial cell–specific growth factors13 that appear to be natural mediators of angiogenesis following hypoxia.14 The VEGF family appears preferable for use in angiogenic therapy, since several members of this family, unlike the FGF family, are secreted angiogenic polypeptides10 that stimulate endothelial cells but do not stimulate smooth muscle cell growth.4 12
Treatment with systemic angiogenic factors would not be an appropriate therapy, because tissue ischemia is a localized process. Therefore, a focal therapeutic approach needs to be developed. Focal genetic modification of tissue can be achieved by ex vivo transduction and transplantation or by direct in vivo gene delivery.15 Viral vectors based on adenovirus or herpes simplex virus type 1 (HSV-1) are among the most efficient vehicles for gene transfer into nondividing cells,16 17 18 19 20 and each has been demonstrated to be capable of focal gene delivery in vivo.16 17 18 19 20 In the present study, we describe the construction and use of an HSV-1 amplicon vector that transduces the angiogenic growth factor, human VEGF-165 (hVEGF-165). Our results demonstrate that an amplicon vector, HSVhvegf, can transduce and express VEGF in cultured fibroblasts, induce endothelial cell proliferation, and, when used in an ex vivo protocol, stimulate angiogenesis in animals.
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Materials and Methods |
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Cell Lines
NIH 3T3 cells and BLK-CL4 cells (fibroblasts derived from C57BL/6 mice) were obtained from the American Type Culture Collection. Bovine capillary endothelial (BCE) cells (from adrenal gland) were kindly provided by Drs Anne Marie Schmidt and Jerry Brett, Columbia College of Physicians and Surgeons, New York, NY.
Cloning of hVEGF-165
DNA from 10 subfractions of a fetal human brain cDNA library in
gt11 each representing 
50 000 independent clones were
amplified by polymerase chain reaction (PCR) using oligonucleotides
MB-4 (5'-GGTCCGGCCTCCGAATTCATGAACTTTCGTCT-3') and MB-5
(5'-GGGAAGCTCCTTCCTGCAGCCCGGCTCACCG-3'), which introduce an
EcoRI and a Pst I site 5' and 3' of the VEGF
coding region. PCR was carried using Taq polymerase and the
manufacturer's buffer (Boehringer), to which formamide was added to
attain a concentration of 5%. The PCR reaction was carried out for 30
cycles consisting of 1 minute at 92°C, 2 minutes at 50°C, and 3
minutes at 72°C. The 650-bp PCR product was digested with
EcoRI and Pst I, purified, and subcloned into
pSP64 (Promega). The cloned hVEGF-165 was subsequently subcloned in
pBluescript KS-1 (Stratagene) and fully sequenced; the plasmid obtained
was denominated pBS-hVEGF-165.
Plasmid Construction
For expression in mammalian cells, pBS-hVEGF-165 was digested
with EcoRI, and the VEGF fragment was subcloned into the
EcoRI site of pEE1421 to generate
pEE14-hVEGF-165, in which VEGF-165 expression was under the
transcriptional control of the cytomegalovirus (CMV) promoter. For
construction of the HSV-1 amplicon vector, pBS-hVEGF-165 was digested
with HindIII–Xba I and ligated into pHSV-Puc,
which was digested with the same enzymes to produce pHSVhvegf-165,
in which VEGF-165 is under the transcriptional control of the HSV
immediate-early (IE) 4/5 promoter. As a control for HSV gene transfer
experiments, we used HSVlac, a virus expressing the lac Z
gene of Escherichia coli instead of VEGF.22
Packaging of HSV-1 Vectors Into HSV-1 Virions
pHSVhvegf-165 was packaged into HSV-1 particles by using a
modified deletion mutant packaging system.23 24 The titers
of the virus stocks were as follows: 5x107 infectious
particles of HSVhvegf per milliliter and 107 plaque-forming
units (pfu) of D30EBA (helper virus) and 1.5x108
infectious particles of HSVlac and 2x107 pfu of D30EBA.
For the ex vivo angiogenesis experiments, the virus was purified and
concentrated 5- to 10-fold.20
Expression of VEGF-165 in Mammalian Cells and Partial Purification
COS-7 cells were seeded at a density of 1.25x106
cells per 35-mm plate in DMEM–10% fetal calf serum (FCS) and
incubated overnight. The cells were transfected with 12-µL
lipofectamine (GIBCO-BRL) and 2 µg DNA according to the
manufacturer's directions. After 5 hours, the transfection mixture was
removed and replaced with DMEM–10% FCS. Conditioned medium was
collected 48 and 96 hours after transfection. To partially purify the
VEGF, 60 mL of pooled conditioned medium was adjusted to 0.3 mol/L NaCl
and loaded onto a Hi-Trap heparin (Pharmacia) affinity column with an
FPLC pump (Pharmacia), washed with 10 column volumes of 10 mmol/L Tris
buffer (pH 7.5) and 0.4 mol/L NaCl, and eluted with the same buffer
containing 0.6, 0.8, or 1.0 mol/L NaCl. Fractions were analyzed for
immunoreactivity by Western blot and for bioactivity by a mitogenesis
assay on BCE cells.
HSV Gene Transfer Into Fibroblasts in Culture
NIH 3T3 cells were plated at a density of 5.0x105
cells per 35-mm well in DMEM–10% FCS. The day after plating, cells
were transduced with 0.5 to 1.0x106 infectious particles
of HSV-1 stock. After 5 hours of incubation at 37°C, the virus was
aspirated, and the cells were washed three times and incubated with
DMEM–10% FCS. Analysis of conditioned media and cells was performed
24 hours later.
Endothelial Cell Mitogenicity Bioassay
Mitogenic assays were performed on BCE cells. BCE cells were
maintained in MEM supplemented with 10% FCS and antibiotics (1%
penicillin and 1% streptomycin). For proliferation assays, the cells
were split 1:3 in DMEM supplemented with 10% FCS and 1 ng/mL basic FGF
(R & D Systems). Cells were seeded in 12-well plates at a density of
1x104 cells per well. Medium to be assayed for angiogenic
activity was added to cells 2 hours after seeding. After 5 days, cells
were trypsinized and counted with an electronic counter (Coulter Corp).
PCR Detection of VEGF Expression
NIH 3T3 fibroblasts were harvested 24 hours after transduction
by placing the cell pellet in 1 mL of Trizol reagent (BRL-GIBCO), and
total RNA was extracted by following the manufacturer's procedure.
cDNA synthesis was carried out in 20 µL of PCR buffer (Boehringer) by
using 2 µg of total RNA and 50 U avian myeloblastosis virus (AMV)
reverse transcriptase (Boehringer) in the presence of 1 mmol/L dNTPs,
20 U RNAsin (Promega), and 5 mmol/L random hexamers for 10 minutes at
23°C, 45 minutes at 42°C, and 5 minutes at 95°C. PCR was
performed by using a sense primer, HF-99
(5'-TCCGACGACAGAAACCCACCGGTC-3'), corresponding to the 5' untranslated
region of the HSV-1 IE 4/5 promoter, and an antisense primer, MB-7
(5'-ATCCGCATAATCTGCATG-3'), corresponding to the VEGF antisense strand
along with Taq DNA polymerase and its manufacturer's buffer
(Boehringer), to which formamide was added to attain a concentration of
5%. The PCR reaction was carried out for 30 cycles consisting of 1
minute at 92°C, 2 minutes at 45°C, and 3 minutes at 72°C.
Immunodetection of VEGF
Samples (conditioned media or matrigel plugs) were dissolved by
mixing with 1/4 volume of 5x denaturing/loading buffer (1x buffer
contains Tris [pH 7.4], 1% sodium dodecyl sulfate [SDS], and 0.2%
ß-mercaptoethanol) and heating at 100°C for 5 minutes. Samples were
electrophoresed in SDS–15% polyacrylamide gel and electroblotted onto
nitrocellulose. The blot was blocked with 3% nonfat dry milk–2%
glycine–Tris-buffered saline and incubated with a 1:250 dilution of a
rabbit polyclonal antibody raised against a peptide encompassing amino
acids 4 to 24 of mature VEGF (anti-VEGF). The blots were developed
either with the Vectastain-peroxidase kit (Vector Laboratories) or the
ECL kit (Amersham). Autoradiograms of Western blot were scanned for
VEGF quantification by using a Laser Ultroscan (LKB).
Matrigel Angiogenesis Assay
The angiogenesis assay was similar to that described
previously.25 26 Briefly, confluent cultures of BLK-CL4
cells were dislodged with cell dissociation buffer (GIBCO-BRL),
recovered by centrifugation, and resuspended in DMEM–10% FCS,
immediately mixed with purified stocks of defective HSV-1, and
incubated at room temperature for 30 minutes. The infected cultures
were cooled on ice for 1 minute and carefully mixed with liquid
matrigel (Collaborative Biomedical) maintained on ice. Part of the
matrigel cell suspension was seeded on 24-well plates (200 µL per
well), and the remainder was injected subcutaneously into 8- to
10-week-old female C57BL/6 female mice (700 µL per mouse). One week
later, the mice were killed, and the matrigel pellet was recovered and
processed for direct hemoglobin quantification by the Drabkin
method,25 histological examination (Masson-Trichrome
staining), and Western blot. All the procedures involving the use of
mice were performed in accordance with institutional guidelines.
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Results |
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Cloning and Characterization of VEGF-165
VEGF exists in four forms: VEGF-121, 165, 190, and 201. Only the two smaller forms are secreted into the medium; the high molecular weight forms are cell-associated.27 We cloned the cDNA for human VEGF-165 by PCR amplification of a human fetal brain cDNA library. The hVEGF-165 clone was fully sequenced and further characterized by immunoblotting and bioassay of the protein expressed by transfected mammalian cells. COS-7 cells were transfected with pEE14–hVEGF-165, a mammalian VEGF expression vector, and medium conditioned by the cells was collected. Samples were assayed for biological activity in a BCE mitogenesis assay. As shown in Fig 1A
, media conditioned by
pEE14–hVEGF-165–transfected cells but not control vector
contained biologically active VEGF. The secreted bioactive material
produced by transfected COS-7 cells and partially purified by heparin
affinity chromatography was intact VEGF, as shown in Fig 1B.
SDS–polyacrylamide gel electrophoresis indicated that under reducing
conditions the major immunoreactive protein had an apparent molecular
mass of 21 kD (lanes 2 and 3). The amount of VEGF was estimated by
densitometry, and the EC50 of the pooled fractions for BCEs
was determined (Fig 1C).
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Expression of VEGF-165 From a Defective HSV-1 Vector
The VEGF-165 cDNA was subcloned into pHSV-PrPuc, generating
pHSVhvegf (Fig 2), in which the VEGF cDNA is under the
transcriptional control of the HSV IE 4/5 promoter. A vector that
expresses E coli ß-galactosidase, HSVlac, was used as a
control.22 NIH 3T3 fibroblasts transduced with HSVhvegf
virus secreted high amounts of biologically active VEGF-165 into the
media (Fig 3A). In contrast, cells transduced with
HSVlac did not secrete a mitogenic protein (Fig 3A). To confirm that
transduced NIH 3T3 cells expressed the virally encoded VEGF gene, we
performed a reverse transcriptase PCR assay on RNA extracted from
HSVhvegf- and HSVlac-transduced cells. As shown in Fig 3B, when RNA
extracted from HSVhvegf-transduced cells (lanes 2 and 3) but not
HSVlac-transduced cells (lane 4) was converted to cDNA and amplified,
it yielded the expected 402-bp product. When reverse transcriptase was
omitted from RNA samples extracted from HSVhvegf-transduced cells
(lanes 6 and 7), no PCR products were observed, demonstrating that the
template for the reaction was RNA.
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) or HSVhvegf (
). B, Reverse transcriptase polymerase
chain reaction of total RNA using a sense herpes simplex virus (HSV)
primer and an antisense VEGF-165 primer. Lanes are as follows: 1,
nontransduced cells; 2 and 3, cells transduced with 0.5 to
1.0x106 IP HSVhvegf; 4, cells transduced with
1.0x106 IP HSVlac; M, molecular weight markers (100-bp DNA
ladder, BRL); and 5, 6, 7, and 8, reactions run in a manner identical
to those in lanes 1, 2, 3, and 4, respectively, without added reverse
transcriptase. C, Western blot from medium conditioned by
HSVhvegf-transduced NIH 3T3 cells reacted with anti-VEGF antibody.
Lanes are as follows: 1, mock-transduced; 2, transduced with HSVhvegf;
3, transduced with HSVlac; 4, blank; 5, VEGF from COS-7–transfected
cells (heparin affinity pool); and M, molecular mass markers.
VEGF-165 secreted from HSVhvegf-transduced cells was detected by
Western blotting of conditioned media (Fig 3C
). Its concentration was
estimated by comparison with standards on Western blots at 1 µg/mL
(data not shown). These data show that HSVhvegf can transduce mouse
fibroblasts and direct the synthesis and secretion of high levels of
bioactive VEGF.
In Vivo Angiogenesis Assay
To test whether cells transduced by HSVhvegf could elicit an
angiogenic response, we sought an ex vivo approach. One such approach
involves the use of a basement membrane extract, "matrigel," to
allow easy quantification of the angiogenic
response.25 26 28 29 In initial experiments, fibroblasts
were transduced with HSVhvegf or control HSVlac virus and mixed with
liquid matrigel, plated in tissue culture wells, and subsequently
assayed for the secretion of VEGF and viability. The results indicate a
sustained expression and release of VEGF from the gelled matrigel into
the conditioned medium (Fig 4A) for the duration of
the 1-week experiment (Fig 4C). Analysis of HSVlac-transduced
fibroblasts by X-gal staining showed that cells remained within the
gelled matrix, appeared healthy, and expressed ß-galactosidase at the
end of the week (Fig 4B); these cells didn't express VEGF as shown by
Western blot analysis (data not shown). The viability of cells that
were transduced by HSVhvegf or HSVlac or that were mock-infected, as
determined by a cytotoxicity assay, was similar throughout the entire
week (data not shown). This indicates that neither infection with the
different recombinants or helper viruses nor the expression of the VEGF
or lacZ transgene affected cell viability. These in vitro studies
suggested the feasibility of an ex vivo study.
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Liquid matrigel containing either HSVhvegf- or HSVlac-transduced
BLK-CL4 fibroblasts was injected subcutaneously into syngeneic C57BL/6
mice. Once injected, the liquid matrigel solidifies and is an excellent
support for angiogenic growth factor–dependent neovessel
formation.25 26 28 29 After 1 week, animals were killed
and angiogenesis was assessed. In Fig 5A,
representative lower power micrographs of matrigel plugs from
HSVhvegf and HSVlac are shown. Marked angiogenesis is observed only in
the matrigel plug containing HSVhvegf-transduced cells. Quantitative
measurement of hemoglobin content in these matrigel plugs (Fig 5B)
indicates a significant increase in HSVhvegf plugs compared with HSVlac
plugs at both multiplicities of infection. Moreover, analysis of
matrigel plugs directly by Western blotting (Fig 5C) shows the marked
expression of VEGF at the second and the seventh day after subcutaneous
injection. No VEGF expression was observed in HSVlac plugs, ruling out
the possibility of a reactive induction and expression of murine VEGF.
Although gross visual inspection (Fig 5A) and hemoglobin content (Fig 5B) suggested a marked increase in apparent capillary content of
HSVhvegf plugs, we further examined plugs histologically, looking
directly for new capillary formation. As shown in Fig 6,
plugs taken from HSVhvegf-transduced animals contained many capillaries
discernible at both low (Fig 6A) and high magnification (Fig 6C). At
high power, alveolar islands of endothelia were seen with budding
proliferation and capillary neovascularization (Fig 6C). Virtually no
capillaries were observed in plugs containing HSVlac-transduced cells
(Fig 6B and 6D). At high power, small focal clusters of one to eight
cells were seen with no vessels or red blood cells (Fig 6D). These
results demonstrate that HSVhvegf can be used in an ex vivo
paradigm to stimulate marked local angiogenesis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Angiogenic therapy is an experimental therapeutic approach for tissue ischemia. Direct administration of an angiogenic growth factor is the simplest method, but it has the potential problem of producing undesirable neovascularization at sites other than those under treatment. Gene transfer strategies have the potential to focally restrict expression of an angiogenic factor and therefore may eliminate the problem associated with systemic delivery. Gene therapy approaches could involve the ex vivo transduction of cells and subsequent transplantation to the disease site and/or the direct introduction of a viral vector that will efficiently transduce the ischemic tissue with an angiogenic gene. We have developed defective HSV amplicon vectors that are suitable for both ex vivo20 30 31 and in vivo20 gene transfer. In the present study, we used an HSV vector that transduces the gene for the potent secreted angiogenic factor VEGF-165. Our data demonstrate that this vector, HSVhvegf, is effective in gene transfer and directs the expression of biologically active VEGF in cultured cells and in animals. When this vector is used in an ex vivo paradigm, it can dramatically stimulate focal angiogenesis.
Recent angiogenesis studies have used growth factors from the FGF and VEGF families.3 4 5 6 15 Although VEGF-165 is somewhat less mitogenic than FGF for endothelial cells, its high specificity for this cell type makes VEGF the preferred factor when only an angiogenic response is sought. FGF also promotes vascular smooth muscle cell proliferation, which in the setting of gene therapy for vascular insufficiency could produce an undesirable atherogenic effect.4 15 32 On the basis of these considerations, we selected VEGF-165 as our candidate angiogenic factor.
Rather than directly inject the viral vector into an organ to induce angiogenesis, we used an ex vivo approach in which transduced fibroblasts were resuspended in a basement membrane extract, matrigel. The use of this matrigel system could reduce potential host-transduced cell interactions that could alter an angiogenic response, and it allows careful evaluation of a number of experimental parameters.25 26 Although the present study clearly demonstrated angiogenesis in animals with HSVhvegf matrigel plugs compared with plugs containing HSVlac-transduced cells, we cannot exclude the remote possibility that secretion of hVEGF from a matrigel plug produced an immune response that augmented local angiogenesis. However, histological analysis did not disclose an inflammatory cell response, and a similar angiogenic response was observed in XID nude mice injected with NIH 3T3 fibroblasts transduced by HSVhvegf (E.A. Mesri, unpublished data). This lack of immune response is consistent with the high degree of homology between murine and human VEGF.33
Effective angiogenesis therapy will likely require high levels of
angiogenic growth factors in order to trigger
neovascularization.4 Once capillaries form and
collateralize, they presumably do not require ongoing angiogenic factor
expression.34 Therefore, a system that produces high-level
short-term expression using a strong viral promoter may be preferable
to one that achieves low-level sustained expression from a
tissue-specific promoter. Time-course experiments (Fig 4) with
HSVhvegf-transduced cells revealed that VEGF expression was between 200
and 400 ng/mL for at least 1 week. This level of expression likely
accounted for the robust angiogenic response observed in vivo (Figs 5 and 6).
Experimental therapeutic targets for angiogenic gene therapy include those currently being approached by direct administration of angiogenic growth factors themselves, such as myocardium3 4 and chronic ischemic limbs.5 6 Given that HSV amplicon vectors can efficiently transduce nondividing cardiocytes and endothelial cells (H.J. Federoff, unpublished data), direct gene transfer approaches for these clinical problems are feasible. Moreover, the HSV amplicon vector can be engineered to contain regulatory elements that are responsive to administered pharmaceuticals and possibly to endogenous regulators (B. Lu and H.J. Federoff, unpublished data). Particularly attractive in this regard is the inclusion of hypoxia-inducible cis elements35 in an HSV amplicon vector such that augmented expression of its angiogenic factor would be subject to regulation by local tissue oxygen content. In the present study, we have demonstrated that the HSV amplicon vector has potential as a vehicle for the induction of angiogenesis. Evaluation of its efficacy in various disease models will be necessary to optimize parameters most useful for therapy.
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Acknowledgments |
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This study was supported in part by grants 192111 (Dr Brownlee) and 192214 (Dr Federoff) from the Juvenile Diabetes Foundation. We are very grateful to Diane Edelstein for the excellent work with the viral stocks, Drs Ida Giardino and Ying-Xian Liu for useful advice and continuous support, and Dr J.J. Steimberg for his valuable help.
Received August 19, 1994; accepted November 3, 1994.
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