Am J Physiol Heart Circ Physiol 285: H1123-H1131, 2003;
doi:10.1152/ajpheart.00103.2003
0363-6135/03 $5.00
Targeting 
v
3 and 51 for gene delivery to proliferating VSMCs: synergistic effect of TGF-1
Jian-Mei Li,1,2
Lampson M. Fan,1
Ajay Shah,1 and
Gavin Brooks3
Departments of 1Cardiology and
2Clinical Sciences, Institute of Liver Studies, King's
College London, London SE5 9PJ; and 3Cardiovascular
Research Group, School of Animal and Microbial Sciences, The University of
Reading, Reading, Berkshire RG6 6AJ, United Kingdom
Submitted 3 February 2003
; accepted in final form 30 April 2003
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ABSTRACT
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TGF-
1 levels increase after vascular injury and promote vascular
smooth muscle cell (VSMC) proliferation. We define a nonviral gene delivery
system that targets 
v3 and 51 integrins that are
expressed on proliferating VSMCs and strongly induced by TGF-1. A
15-amino acid RGDNP-containing peptide from American Pit Viper venom was
linked to a Lys(16) peptide as vector (molossin vector) and complexed with
Lipofectamine or fusogenic peptide for delivery of luciferase or
-galactosidase reporter genes to primary cultures of human, rabbit, and
rat VSMCs. Preincubation of VSMCs with TGF-1 for 24 h, but not with
PDGF-BB, interferon-
, TNF-, nor PMA, increased v3
and 51 expressions on VSMCs and enhanced gene delivery of
molossin vector. Thus -galactosidase activity increased from 35 ±
5% (controls) to 75 ± 5% after TGF-1 treatment, and luciferase
activity increased fourfold over control values. Potential use of this system
in vessel bypass surgery was examined in an ex vivo rat aortic organ culture
model after endothelial damage. Molossin vector system delivered
-galactosidase to VSMCs in the vessel wall that remained for up to 12
days posttransfection. The molossin vector system, when combined with
TGF-1, enhances gene delivery to proliferating VSMCs and might have
clinical applications for certain vasculoproliferative diseases.
integrin; peptide vector; molossin rector
PROLIFERATION AND MIGRATION of vascular smooth muscle cells
(VSMCs) play major roles in the development of intimal hyperplasia and are
crucial events that lead to chronic allograft rejection, postangioplasty
restenosis and in-stent stenosis
(1,
15). The major rationale for
early genetic intervention in the treatment of these disorders is to deliver
functional copies of genes that control proliferation and/or migration
specifically into VSMCs. However, those nonviral DNA vectors that currently
are available have low levels of efficiency and lack specificity, whereas
viral vectors, such as retroviral or adenoviral vectors, display high gene
transfer efficiencies but suffer from immunogenicity, potential oncogenic
risks when used in the clinical setting, and they are not specific for
proliferating and migrating VSMCs
(20). Therefore, a nonviral
DNA vector system, with no toxicity but high efficiency and selectivity for
proliferating and migrating VSMCs, would greatly enhance the prospect of gene
therapy for treating vasculoproliferative diseases.
Integrins are heterodimeric transmembrane receptors consisting of
noncovalently linked - and -subunits that mediate many cellular
responses in VSMCs and other cell types, including differentiation,
proliferation and migration
(11,
23,
27). Furthermore,
51 and v3 have been implicated in pathological
conditions such as atherosclerosis, restenosis after angioplasty and
constrictive vascular remodeling after injury
(25). Vascular injury induces
51 integrin expression exclusively in proliferating VSMCs at the
luminal surface of the neointima
(17), and VSMC invasion from
the tunica media to the intima has been shown to be dependent on
v3 integrin expression
(13). Interestingly,
integrin-mediated cell entry is exploited by a number of intracellular
pathogens including adenovirus and encovirus
(5). The widespread use of this
means of cell entry in nature suggests that integrin-binding ligands might be
suitable moieties for the development of receptor-mediated gene delivery
vectors (2,
10).
TGF-1 has profound autocrine and paracrine biological effects on
VSMCs and on neointima formation after allograft transplantation and
angioplasty (4,
28). TGF-1 also
contributes to arterial remodeling after injury by stimulating VSMC adhesion
molecule synthesis at sites of vascular damage
(8) and induces 3 and
5 integrin expression in VSMCs
(3,
12). Given the important
implications of TGF- in the pathogenesis of local occlusive vascular
diseases, a selective gene delivery system, which is enhanced by a local
effect from elevated levels of TGF-1 in proliferating and migrating
VSMCs, would be advantageous.
We have used a nonviral gene delivery system consisting of a 15-amino acid
peptide derived from the venom of the American pit viper (Crotalus
molossus) linked to 16 consecutive lysines, to permit electrostatic
binding of DNA, as a vector (molossin vector)
(6,
14,
24). Molossin vector contains
an RGDNP motif that displays high affinities toward v3 and
51 integrins (as opposed to the RGDW motif present in other pit
viper venoms) (22,
31). In the present study, we
have used molossin vector with Lipofectamine or a fusogenic peptide to
introduce genes into proliferating VSMCs pretreated with TGF-1. We also
tested the potential application of the molossin vector system to deliver
genes into the vessel wall in an aortic organ culture model.
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MATERIALS AND METHODS
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Materials. PMA, human recombinant TGF-1, human recombinant
TNF-, human recombinant PDGF-BB, and human recombinant
interferon- (INF-) were from Sigma (St. Louis, MO). Mouse IgG1
monoclonal antibody to molossin peptide (LC2–64) was raised as described
previously (14). Mouse IgG1
antibodies to human v3 integrin (CD49e) and 51
integrin (CD51/CD61) were from Serotec (Bicester; Oxon, UK). Mouse IgG2a
antibody to -smooth muscle actin and mouse monoclonal antibody to
-galactosidase (-gal) were from Sigma.
Peptides. Molossin peptide vector, consisting of 16 lysines at the
amino terminus
NH2-(Lys)16-Ile-Cys-Arg-Arg-Ala-Arg-Gly-Asp-Asn-Pro-Asp-Asp-Arg-Cys-Thr-COOH,
was synthesized, purified and cyclized by Cambridge Research Biochemicals
(Zeneca-CRB; Northwich, Cheshire, UK). HPLC confirmed purity as 96.7%. The
20-amino acid fusogenic peptide of influenza virus
(NH2-Gly-Leu-Phe-Glu-Ala-Leu-Leu-Glu-Leu-Leu-Glu-Ser-Leu-Trp-Glu-Leu-Leu-Leu-Glu-Ala-COOH)
was synthesized and purified by Zinsser Analytic (Maidenhead, Berkshire, UK).
Purity was >90%. Peptides were dissolved in 0.15 M NaCl at stock
concentrations of 0.1 mg/ml and then stored in small aliquots at
–35°C.
Primary culture of VSMCs. Normal primary cultures of human aortic
VSMCs (batch F-14579) were obtained from American Type Culture Collection
(Manassas, VA) and maintained in Ham's F-12K medium supplemented with 10%
fetal calf serum (FCS), 2 mM L-glutamine, 10 µg/ml insulin (Life
Technologies, Paisley, UK), 1 µg/ml transferrin (Sigma), 10 ng/ml sodium
selenite (Sigma), and 20 µg/ml endothelial cell growth supplement (Sigma).
Rat and rabbit VSMCs were isolated from thoracic aortas as described
previously (14). Cells from
all three species were used between passages 2 and 6 and
were in the synthetic (proliferative) stage of growth. In all cases, >99%
of cells stained positive for -smooth muscle actin.
Formation of transfection complexes and transfection of VSMCs. The
methods and concentrations of each component required to form transfection
complexes have been examined and optimized previously
(14,
24,
30,
31). A molossin
(fuso-molossin)-vector/DNA/Lipofectamine ratio of 5:1:2 was used to achieve
maximum transfection efficiency
(14). VSMCs (1 x
105/well in 24-well plates) were seeded in appropriate culture
medium the day before transfection. Cells then were cultured for appropriate
periods of time in the presence or absence of TGF-1 (1–20
µg/ml). Cells were transfected with 0.4 ml of DNA complex for 3–4 h
after which an equal volume of culture medium containing 20% FCS was added
followed by overnight culture. Medium then was replaced with fresh medium.
Three days after initial transfection, cells were analyzed for expression of
reporter genes.
Reporter gene assays. -Gal and luciferase reporter gene
assays were performed as described previously
(14). For organ transfection,
vessels were cut into 5-mm segments and stained for -gal as described
(14). Vessels were fixed in
10% formalin, wax-embedded, cut into 5-µm sections, and counterstained with
neutral red (to stain nuclei). Luciferase enzyme activity was measured with a
luciferase assay kit (Promega), and results were expressed as relative light
units per milligram of total protein.
Ex vivo transfection of aortic vessel with -gal gene and
immunocytochemistry. Adult male Wistar rats were anesthetized terminally
by injection of pentobarbitone (0.6 mg/g body wt ip). The thoracic aorta was
removed, and the endothelium was injured by three passes with a 3-mm diameter
balloon. After being washed, one end of the vessel was clamped with a small
hemostat and the other end of the aorta was mounted on a small plastic tube
connected to a 1-ml syringe. The liquid in the lumen was decanted, and the
aorta was filled with transfection (-gal) complex solution. The aorta
was placed into DMEM and incubated at room temperature for 10 min after which
both ends of the aorta were trimmed off and the remaining vessel cut into 1-cm
pieces. Three pieces per well were placed into 6-well plates and incubated in
3 ml of transfection complex solution for 4 h after which an equal volume of
medium containing 20% FCS was added overnight. The medium was then replaced
with fresh culture medium. Vessels were cultured for a further 3–14
days, and -gal expression in the vessel wall was examined by
histochemical staining or immunofluorescence on frozen tissue sections as
previously described (14). All
animals were housed and cared for in accordance with current UK Home Office
Guidelines. All protocols adhered to APS's "Guiding Principles in the
Care and Use of Animals."
Statistical analysis. Quantitative data are shown as means
± SD unless otherwise stated. Differences in mean measurements among
experimental groups were tested by ANOVA, followed by a Student's
t-test with a Bonferroni correction, and differences were considered
significant at the 95% level.
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RESULTS
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Effect of TGF-1 on 51
and v3 integrin expression in human VSMCs.
Human aortic VSMCs were used to assess the effect of TGF-1 on
51 and v3 integrin expressions. Cells were
pretreated with or without TGF-1 for 24 h and examined for the
expression of 51 and v3 integrins by flow cytometric
analysis. In the absence of TGF-1, VSMCs expressed basal levels of
51 and v3 integrins
(Fig. 1). However, after
treatment with TGF-1, VSMCs increased 51 and
v3 integrin expressions resulting in a right shift of the flow
cytometry profile. These results are in accordance with previous reports that
TGF-1 induces 5 and 3 integrin expression in vascular cells
(3,
12) and suggest that use of a
molossin vector, which is known to target 51 and v3
integrins, might be more effective as a gene delivery system in VSMCs when
under the influence of TGF-1.
Effect of TGF-1 on lipo-molossin- or
fuso-molossin-mediated gene transfection. The molossin vector previously
has been used in cultured VSMCs
(14) and in other cell types
and tissues (24,
31), and the system needs
Lipofectamine or fusogenic peptide to assist endocytic exit
(14,
30). To determine the
transfection efficiency before and after TGF-1 pretreatment of VSMCs, we
used the -gal and GFP reporter genes.
Figure 2 shows that
transfection of rat VSMCs with the -gal gene in combination with the
Lys(16) alone failed to yield any -gal expression
(Fig. 2A), whereas
molossin vector (Fig.
2B) or Lipofectamine
(Fig. 2C), when used
alone, enabled 
2–3% transfection efficiency.
Figure 2D shows that
cells transfected with -gal in combination with Lipofectamine-Lys(16)
led to a small increase in transfection efficiency (5 ± 2%) compared
with Lipofectamine (Fig.
2C). There was no difference in the level of transfection
between cells treated with or without TGF-1. Interestingly, when
molossin vector was used in combination with Lipofectamine (lipo-molossin)
(Fig. 2E),
transfection efficiency was increased to 75 ± 5% in rat VSMCs
pretreated with TGF-1 compared with 35 ± 5% in cells without
TGF-1 pretreatment (Fig.
2F). Thus the effect was dependent on the presence of the
integrin targeting moiety of the vector, because TGF-1 pretreatment did
not improve gene delivery when a Lys(16) control peptide was used with
Lipofectamine (Fig.
2D). Similarly, fusogenic peptide when used alone
(Fig. 2G) displayed no
evidence of transfection as determined by measuring GFP expression regardless
of whether cells were pretreated with or without TGF-1. However, when
molossin vector formed complexes with fusogenic peptide (fuso-molossin) it
successfully delivered the GFP reporter gene to human VSMCs. The transfection
efficiency of fuso-molossin was 40 ± 5% in TGF-1 pretreated human
VSMCs (Fig. 2H)
compared with 25 ± 5% in the same cells without TGF-1
pretreatment (data not shown). Thus pretreatment of cells with 10 ng/ml
TGF-1 for 24 h before transfection increases transfection efficiency of
both lipo-molossin and fuso-molossin vectors twofold when compared with cells
not pretreated with TGF-1.
Using neutralizing antibodies to 51 and v3
integrins, we demonstrated that lipo-molossin targets VSMCs. Thus
Fig. 3 shows that incubation of
cells with antibodies to 51 or v3 for 1 h before
transfection significantly (P < 0.03) inhibited molossin vector
delivery of the luciferase gene (as measured by luciferase activity) compared
with cells incubated with medium only. The combination of the two antibodies
inhibited luciferase activity by 85% without TGF-1 and 89% with
TGF-1. These results suggest that molossin vector targets
51 and v3 integrins and that it might be more
effective as a gene delivery system in VSMCs when under the influence of
TGF-1.
We also used lipo-molossin to deliver the luciferase reporter gene into
human aortic VSMCs pretreated with different concentrations of TGF- for
24 h or at a fixed concentration (10 ng/ml) but during variable periods
(Fig. 4A).
Pretreatment of cells with TGF-1 significantly increased luciferase
reporter gene expression. Thus the luciferase activity in TGF-1 (10
ng/ml) pretreated VSMCs was approximately sixfold of that present in cells
without TGF-1 pretreatment (Fig.
4A, top). Therefore, 10 ng/ml of TGF-1 was
used in all subsequent experiments. The effect of TGF-1 on transfection
efficiency into VSMCs was time dependent because the maximal effect, which
produced approximately sevenfold greater luciferase activity than the
activities detected in cells without TGF-1 pretreatment, was observed 24
h after pretreatment (Fig.
4B, bottom). The effect of enhancing molossin
vector gene delivery to VSMCs is TGF-1 specific because pretreatment of
VSMCs with PDGF-BB (10 ng/ml), TNF- (100 U/ml), INF- (100 U/ml),
or the PKC activator PMA (100 ng/ml) for 24 h had no effect on lipo-molossin
gene delivery (Fig.
4B). The effect of TGF-1 was further examined in
primary VSMC cultures derived from human, rabbit, and rat. Compared with cells
without TGF-1 treatment, TGF-1 pretreatment significantly
increased the efficiency of gene delivery of both fuso-molossin and
lipo-molossin vectors to all three species of VSMCs (P < 0.03)
(Fig. 4C).
Interestingly, lipo-molossin gene delivery was greater than that achieved with
fuso-molossin in these studies. One reason for this might involve an increased
binding of transfection complex to the cell surface and better endocytic
escape of molossin vector/DNA with Lipofectamine. Finally,
Fig. 4D shows that
TGF-1 enhances molossin vector delivery of the luciferase gene in human
VSMCs 20-fold more selectively than in some other cell types, including
hepatocytes and fibroblasts.
Ex vivo gene delivery to VSMC in the vessel wall using
lipo-molossin. Figure 5
illustrates that molossin vector bound effectively to medial VSMCs
(Fig. 5B), whereas
control tissue, which had not been incubated with molossin vector
(Fig. 5A), showed
limited background staining.
We then examined the potential application of this gene transfer technology
to deliver genes into VSMCs in the vessel wall in an ex vivo model of rat
aortic organ culture. The -gal reporter gene was employed to assess the
efficiency of lipo-molossin and was visualized by immunohistochemical staining
(Fig. 5,
C–E). Thus control vessels transfected
with lipo-molossin alone (Fig.
5C) or with Lipofectamine plus -gal DNA alone
(Fig. 5D) showed no
evidence of -gal expression. In contrast, vessels transfected with
-gal DNA plus lipo-molossin showed -gal expression (by X-gal
staining) mainly in medial VSMCs (Fig.
5E, blue color). Some -gal expression also was
detected in the junction area between the media and adventitia
(Fig. 5E). Expression
of -gal in the vessel wall also was confirmed by immunofluorescence
staining by using a monoclonal antibody against Escherichia coli
-gal. Expression of -gal was detectable 3 days after initial
transfection, whereas 12 days after transfection, VSMCs had migrated out from
the media layer to form neointima. Interestingly, -gal expression was
detectable in neointima cells (Fig.
6). To quantitatively measure the gene delivery efficiency in rat
aortic vessels, we performed a series of luciferase activity studies
(summarized in Table 1).
Lipo-molossin vector enhanced luciferase activity seven- and fourfold when
compared with enzyme levels measured in the presence of Lipofectamine alone or
Lipofectamine and Lys(16) peptide alone, respectively
(Table 1).
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DISCUSSION
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In this study, we have demonstrated use of a nonviral and highly efficient
gene delivery system utilizing a nontoxic peptide vector that targets
51 and v3 integrins expressed highly on
proliferating VSMCs. The targeting moiety of the vector consists of a 15-amino
acid peptide exhibiting a RGDNP motif that binds preferentially to
51 and v3 integrins
(22) and a DNA binding moiety,
comprising 16 consecutive lysine residues, which promotes electrostatic
binding of DNA. RGD is an integrin recognition sequence that is particularly
effective in those adhesion proteins that bind to the 1, -3, and -5
family of integrins (11,
21). However, internalized DNA
is targeted to lysosomes for destruction
(7) and one important limiting
step for receptor-mediated gene delivery is the exit of DNA from endosomes
(19). In our study, this
process was achieved by inclusion of the cationic lipid, Lipofectamine
(14,
30), or a fusogenic peptide
derived from the hemagglutinin of influenza virus
(9,
19,
30).
A particular attribute of the molossin vector system is that it takes
advantage of the TGF-1 effect on VSMC integrin expression and
proliferation such that a much higher transfection efficiency is achieved in
TGF-1 treated cells. The clinical relevance and importance of this
finding is highlighted by the fact that TGF-1 can be produced by almost
every vascular cell in the vessel wall, and its expression is increased
significantly after vessel injury and/or during other pathological conditions
in which VSMC proliferation forms a part of the pathology
(4,
18). Indeed, in a rat balloon
injury model, high TGF-1 expression levels were found in the injured
vessel wall, which promoted restenosis after angioplasty
(26), whereas local blockage
of TGF-1 using ribozyme oligonucleotides inhibited neointima formation
after vascular injury (29).
One important mechanism responsible for the effects of TGF-1 on VSMC
function and neointima formation is that it increases 5 and 3
integrin expressions (3,
12). Our study confirms that
pretreatment of human VSMCs with TGF-1 significantly increased cellular
expression of 51 and v3 integrins. Furthermore, we
have shown that molossin vector targets 51 and v3
integrins on proliferating human VSMCs. This has formed the foundation of our
studies using the molossin vector for gene transfer into proliferating VSMCs
after vessel injury. The concentration of TGF-1 (1–20 ng/ml)
necessary to increase molossin vector gene delivery is physiologically
relevant. Thus circulating levels of TGF-1 range from 2–10 ng/ml
in healthy individuals (8).
Moreover, at sites of vascular injury, in which vessel wall TGF-1
synthesis is induced and activated platelets release TGF-1, the local
concentration of TGF-1 may commonly reach the levels (>10 ng/ml)
found to be optimal in this study.
In many restrictive vessel cases, a reactive cellular proliferative
response leads to the regrowth of VSMCs locally that reduces lumen diameter
and thereby compromises blood flow. This can occur at sites of anastomoses in
allograft transplants and in bypass grafts and is a major component of
restenosis and in-stent stenosis after angioplasty. A suitable nonviral and
nontoxic vector that mimics the functions of viral proteins for DNA delivery
into proliferating and migrating VSMCs would greatly improve the outcome for
patients after such interventional therapy. With increases in the number of
allograft transplantations and vessel bypass procedures, it would be
advantageous to transfer the gene ex vivo into the isolated grafts before
transferring to the recipient. Indeed, Mann and colleagues
(16) recently transferred
decoy oligonucleotides, which target the E2F transcription factor family, to
veins ex vivo before grafting into patients. This approach led to a reduction
in failure rates of this procedure and improved the patency of grafts and
demonstrates the effectiveness of ex vivo gene therapy for certain vascular
diseases. In the present study, we have shown that successful ex vivo gene
transfer can be achieved with the molossin vector in rat aortic vessels after
injury of the endothelium. Of particular interest was the observation that
molossin vector bound efficiently to VSMCs in aortic vessels denuded of
endothelium (Fig. 5B).
Thus gene delivery occurs readily in proliferating VSMCs in areas of vessel
injury, whereas delivery would likely be minimal or would not occur at all in
uninjured vessels.
In summary, we have demonstrated the use of a nonviral gene delivery system
for proliferating VSMCs both in vitro and ex vivo that is enhanced
significantly in the presence of TGF-1. It is possible that the
combination of molossin vector with TGF-1 might not be suitable for all
in vitro transfection procedures (due to unwanted effects from the cytokine).
In such cases, the use of integrin-activating agents, e.g., activating
antibodies in place of TGF-1, might prove more preferable. However, the
current approach certainly is advantageous for ex vivo/in vivo gene delivery
in areas in which TGF-1 levels are raised naturally, e.g., in injured
vessels. The system provides advantages over viral vectors including safety
and a lack of any limit on the size of DNA insert that can be packaged. The
whole system contains only three components: molossin vector, DNA, and
fusogenic peptide or cationic lipid. All components are molecularly defined,
easily undergo self-assembly into an active DNA delivery system and the whole
procedure can be achieved within a short time frame, thereby facilitating the
application of this technology within the operating theater during bypass
graft or organ transplantation operations.
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DISCLOSURES
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This study was supported, in part, by the British Heart Foundation, the
National Heart Research Fund, and the Royal Society.
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ACKNOWLEDGMENTS
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We thank John Fabre for helpful discussions.
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FOOTNOTES
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Address for reprint requests and other correspondence: G. Brooks,
Cardiovascular Research Group, School of Animal and Microbial Sciences, PO Box
228, Whiteknights, Reading, Berkshire RG6 6AJ, UK (E-mail:
g.brooks{at}reading.ac.uk).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
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Copyright © 2003 by the American Physiological Society.
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ABSTRACT
MATERIALS AND METHODS
