© 1999 American Heart Association, Inc.
Brief Review |
Vascular Gene Transfer
From Bench to Bedside
From the Division of Cardiovascular Diseases and Internal Medicine (I.J.K., R.D.S., R.S.S.), and the Department of Biochemistry and Molecular Biology (R.D.S.), Mayo Clinic and Mayo Foundation, Rochester, Minnesota.
Correspondence to Iftikhar J. Kullo, MD, Division of Cardiovascular Diseases and Internal Medicine, Mayo Clinic, 200 First Street SW, Rochester, Minn 55905.
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Introduction |
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 Top Introduction Part I: Basic PrinciplesPart II: Therapeutic PotentialReferences |
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Since the initial experiments nearly a decade ago, remarkable progress has been made in the field of vascular gene transfer, and trials with human subjects already are under way. The potential of vascular wall gene transfer as therapy for various vascular diseases is considerable.1 2 The purpose of this review is 2-fold: (1) to delineate the methods and principles underlying vascular gene transfer and (2) to discuss how gene transfer might be used in the treatment of vascular diseases.
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Part I: Basic Principles |
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TopIntroductionPart I: Basic PrinciplesPart II: Therapeutic PotentialReferences |
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An overview of the basic principles of vascular gene transfer provides a perspective of its potential for vascular wall gene therapy. In this section, methods of gene transfer to the vascular wall, shortcomings of the available vectors, and techniques of vector delivery to the vascular bed are discussed.
Methods of Gene Transfer to the Vasculature
As a target organ for gene transfer, the vasculature has several
unique features (Table 1
). The principal
methods of introducing genetic material into the vasculature include a
cell-based approach, ex vivo gene transfer to vessel segments, and in
vivo gene transfer to the vascular wall.
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Cell-Based Gene Transfer
Cell-based gene transfer requires harvesting vascular wall cells,
in vitro transduction, followed by seeding the vessel wall with
transduced cells. Although this strategy has several potential
applications, it is technically difficult (Table 2). Both of the major vascular wall cell
types, the smooth muscle cell (SMC) and the endothelial
cell (EC), can be modified genetically for vascular gene transfer.
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SMCs
In culture conditions, vascular SMCs are robust, do not have
stringent growth requirements, and are transduced relatively easily.
Hence, these cells are ideal for cell-based vascular gene transfer.
Potential applications include modulating local vascular phenomena and
obtaining systemic gene delivery.3 Feasibility of
cell-based vascular gene transfer was demonstrated first by Plautz and
coworkers.4 SMCs transduced in vitro with recombinant
ß-galactosidase were infused into denuded porcine iliofemoral
arteries with use of a double-balloon catheter. Transgene expression in
the arterial segments could be demonstrated up to 11 days
after infusion. Recently, such an approach was used to obtain
biological effects. Overexpression of a tissue inhibitor of
matrix metalloproteinase (TIMP)-1 was achieved by seeding SMCs
transduced with the TIMP-1 vector into injured carotid arteries of
rats. This resulted in a significant decrease in neointimal
hyperplasia in comparison with arteries seeded with
control-vector–transduced cells.5 In the same model,
cell-based overexpression of endothelial nitric oxide
synthase (eNOS) resulted in vascular remodeling and luminal
enlargement.6
Genetically engineered SMCs seeded in a vascular graft may be used for
long-term local and systemic gene therapy.7 Although SMCs
could be used as a biological lining for endovascular stents, bypass
grafts, and left ventricular assist devices, such a lining
may be thrombogenic. Gene transfer of eNOS or
cyclooxygenase-l to SMCs is a possible solution to
this problem. Indeed, expression of recombinant eNOS in
coronary artery SMCs increases NOS enzymatic activity and
cyclic 3'-5'-guanosine monophosphate (cGMP) levels and nitrite
production.8 These findings are similar to those
of Scott-Burden and coworkers,9 although in their study
nitric oxide (NO) production by recombinant eNOS occurred only
in the presence of tetrahydrobiopterin, a cofactor for eNOS. Therefore,
phenotypic changes that occur with subculturing ex vivo may affect the
results of cell-based gene transfer. For example, in eNOS gene
transfer, changes in tetrahydrobiopterin availability and
guanylate cyclase activity may determine functionality of
the transgene. Thus, even after successful gene transfer, eventual
effects may be influenced by the nature of the target cell and biology
of the recombinant protein (Figure).
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The potential of SMC-based gene transfer as a means of systemic gene delivery has been demonstrated in a study in which rat arteries were seeded with SMCs transduced with a retroviral vector encoding a cDNA for erythropoietin.10 An increase in red blood cell mass was noted for up to 11 weeks. Other potential methods of systemic delivery using genetically engineered SMCs include embolization into a microvascular bed11 and incorporation into a synthetic structure supplied by an induced neovasculature.12
ECs
The feasibility of using ECs for cell-based vascular wall gene
therapy was demonstrated in a study in which genetically modified ECs
were transplanted successfully into the arterial wall of a
pig.13 Another study demonstrated transgene expression in
Dacron grafts seeded with autologous genetically modified ECs and
implanted as carotid interposition grafts in dogs.14
Dichek's group15 used the cell-based approach to modify
vascular stents. Sheep ECs expressing human tissue
plasminogen activator were seeded onto
balloon-expandable stents in vitro. Complete coverage of stent surfaces
was achieved after several days in culture, and such stents could be
deployed in a pulsatile flow system with significant retention and
survival of the seeded ECs.16 Furthermore, implantation of
ECs expressing tissue plasminogen activator
into vascular grafts decreased platelet and fibrin formation in a
baboon model of arteriovenous shunt thrombosis.17
Cell-based gene transfer using ECs is a potential means of systemic gene delivery. Genetically modified ECs can become incorporated into the endothelium of a microvascular capillary network.11 The capillary bed of skeletal muscle, for example, could serve as a recipient site for transduced, autologous ECs for systemic gene delivery.11 A notable recent advance is the discovery that putative EC progenitors, or angioblasts, are present in human peripheral blood.18 These cells can be separated on the basis of cell surface antigen expression, and they differentiate into ECs in vitro. In animal models of ischemia, these cells home onto foci of angiogenesis.18 Potentially, such cells could be used as autologous vectors for vascular gene transfer. Angiogenesis at sites of myocardial or skeletal ischemia could be enhanced by transfecting EC progenitors with angiogenic transgenes and introducing them into the circulation.
Ex Vivo Gene Transfer to the Vessel Wall
Ex vivo gene transfer is particularly suited for the genetic
modification of venous or arterial bypass grafts. Such
grafts can be genetically engineered ex vivo with optimized
transfection efficiency and minimization of toxicity. The harvested
vein or artery intended for use as a bypass graft can be transduced ex
vivo by placing it in a vector solution before implantation. Liposomes
as well as viral vectors can be used for transducing vessel segments.
Transgene expression generally is noted in the adventitia and the
endothelium but not in the media.19 The
technique is relatively simple and safe, and bypass grafts genetically
engineered ex vivo currently are undergoing testing in clinical
trials.20
In Vivo Gene Transfer to the Vascular Wall
In vivo vascular gene transfer was performed first in porcine
iliofemoral arteries with use of a double-balloon catheter and
retroviral and plasmid vectors encoding
ß-galactosidase.21 Expression of ß-galactosidase could
be demonstrated for up to 3 months after gene transfer. Since this
landmark study, many studies on vascular wall gene transfer have been
performed with different transgenes in different animal models, using
various delivery techniques and vectors. Some of these studies are
discussed below, using the division of the vascular wall into 3 layers
as a framework. A unique feature of vascular gene transfer is that
expression of genetic material can be localized to a specific layer
because of the presence of elastic laminae that act as barriers to
vectors.22
Gene Transfer to the Endothelium
The endothelium is a large, readily accessible
target organ for genetic modulation. Expression of recombinant proteins
in ECs may be useful in elucidating the pathogenetic mechanisms of
diseases such as hypertension and atherosclerosis and
providing novel therapeutic options for disorders characterized by
endothelial dysfunction and thrombosis. Relatively
endothelium-specific gene transfer is obtained by
intraluminal delivery of vectors, because the internal elastic lamina
acts as a barrier to most vectors. In the carotid artery of the rat, a
concentration of 1010 PFU/mL of an adenoviral
vector yielded relatively efficient gene transfer, with minimal effect
on the arterial phenotype.23 Up to
35% of ECs are transduced by this method.23 So far, few
studies have considered the expression of therapeutically relevant
genes in the endothelium in vivo. In a recent study,
endothelium-specific expression of recombinant eNOS
resulted in altered vascular reactivity.24 Significantly
higher cGMP levels were present in eNOS-transduced arteries, and
contractile responses to phenylephrine were diminished.
Additionally, endothelium-dependent relaxations to
acetylcholine were enhanced significantly in the eNOS-transduced
arteries.
Gene Transfer to the Media
SMCs are the principal cellular component of arteries and veins
and have a key role in various vascular diseases, including
atherosclerosis, restenosis, vein graft
disease, and transplant vasculopathy.25 SMC migration,
proliferation, and matrix secretion are important factors in the
development of neointimal proliferation.25
Therefore, SMCs are an important target of various systemic and local
therapies to limit neointimal formation. Luminal gene
delivery may result in medial SMC transduction if the vector solution
is injected at high pressure or the elastic lamina is disrupted by
balloon angioplasty. Several investigators have demonstrated decreased
neointimal formation by expression of various transgenes in
the media after arterial injury.
Gene Transfer to the Adventitia
Adventitial gene transfer avoids interruption of blood flow,
disruption of the endothelium, and systemic
distribution of the vector.26 Recently, the adventitia has
been shown to participate in remodeling and neointimal
formation after arterial injury.27 28 29 The
adventitia also may influence atherogenesis30 and vascular
hypertrophy secondary to hypertension.31
Therefore, the adventitia is a target for site-specific vascular wall
therapy. Adventitial-specific gene transfer after delivery of
adenoviral vectors into the periarterial sheath has been
demonstrated in different animal models.26 32 Adventitial
delivery of vectors for gene transfer may be achieved easily in large
peripheral arteries by direct application during surgery
and in cerebral arteries by instillation into the cerebrospinal
fluid.33 Adventitial delivery to the coronary
arteries may be possible with novel strategies such as delivery of
vectors to the pericardial space34 or by
percutaneous intervention using special
catheters.35 Gene transfer to the adventitia of
pulmonary arteries is achieved by aerosolization of adenoviral
vectors.36 Expression of recombinant eNOS in the
adventitia of the carotid arteries of rabbits leads to a marked
increase in NOS enzymatic activity and an alteration in vascular
reactivity.32 Delivery of the eNOS vector to the
cerebrospinal fluid in a dog model led to expression of the transgene
in the adventitia of cerebral vessels and enhanced relaxations of the
vessels to bradykinin.37
Vectors for Vascular Gene Transfer
Ideally, a vector for gene transfer should be easily produced,
available in a concentrated form, tissue-specific, nontoxic, and
nonimmunogenic, provide long-term expression, and allow a broad range
of transgene size. Such a vector currently does not exist. A detailed
description of the vectors available for vascular gene transfer is
outside the scope of this review. The advantages and disadvantages of
the commonly used vectors for vascular gene transfer are listed in
Table 3. Technical improvement in vectors
may permit more efficient and cell-specific gene transfer, avoid
immune-inflammatory responses, and maintain gene expression for long
periods. These improvements would be a major step toward realizing the
potential of gene therapy for vascular diseases. Newer vectors for gene
transfer include the adeno-associated virus (AAV) and lentivirus. Like
adenoviral vectors, AAV vectors are able to transduce many different
cell types and do not need active cell proliferation for transduction.
Additionally, these vectors may be less immunogenic and may obtain a
more prolonged transgene expression.41 The usefulness of
AAV vectors in vascular gene delivery has been demonstrated in cultured
SMCs and ECs from various species as well as in vivo delivery to the
arterial wall.42 Lentiviral vectors are able
to infect nondividing cells, overcoming a major drawback of retroviral
vectors.43 Concern about the safety of such vectors led to
attempts to develop multiply- attenuated lentiviral
vectors.44 Vectors based on nonprimate lentiviruses such
as feline immunodeficiency virus are also being
evaluated.45 The major shortcomings of available vectors
and ways to improve these are discussed below.
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Vectors Elicit Inflammation
Viral vectors have the potential to induce an inflammatory-immune
response,46 leading to tissue damage, and a decrease in
the duration of transgene expression, particularly in case of
adenoviral vectors. Adenoviral vectors, from which all transcriptional
coding regions have been deleted (`backbone' vectors), have been
developed to minimize inflammatory or immune responses.47
Immune response to viral vectors could be inhibited by administering
immunosuppressant drugs48 or cotransfection of vectors
expressing immunomodulatory genes.49
Expression of Transgenes May Be Transient
Long-term expression of transgenes may be desirable in certain
situations. This requires chromosomal integration with retroviral
vectors or the stable episomal presence of an adenoviral vector without
the loss of transgene promoter activity. In tissues with high cell
turnover, the expression of transgene after adenoviral-mediated gene
transfer may be shortened by loss of the episomal, nonreplicating
vector genome. In addition, clearance of transduced cells by the host
immune system also limits duration of transgene expression. A decrease
in the activity of the promoter in vivo ("promoter attenuation")
due to incompletely understood mechanisms is also responsible for
decreased transgene expression with time.50 Increased
duration of expression may result from improved vector design or by
modulation of the host defense system, as discussed above.
Vectors Lack Cell or Tissue Specificity
Targeted gene delivery can be accomplished by using either
cell-specific promoters or constructing vectors with defined cell
tropism. Tissue-specific expression can be achieved by using a tissue-
or cell-specific promoter to drive the transgene. Examples of
endothelial-specific promoters include
thrombomodulin,51 von Willebrand
factor,52 and tie-2. The latter is an
endothelial-specific receptor tyrosine kinase essential
for the regulation of vasculogenesis and
remodeling.53 Use of the tie-2 promoter
resulted in reporter gene expression in vascular ECs throughout
embryogenesis and adulthood in mice.54 An
arterial SMC-specific promoter, SM22 alpha, that
activates transcription exclusively in arterial
SMCs has been used to restrict expression of a recombinant adenoviral
vector to SMCs.55
Vectors with a defined cell tropism may be constructed by several different strategies. For viral vectors, one approach for altering tropism is to modify surface proteins that interact with cell surface receptors. This has been demonstrated for retroviral vectors.56 In adenoviral vectors, tissue specificity may be altered by modifying either the fiber or penton capsid proteins that mediate interaction with cell surface receptors.57 58 Plasmid DNA can be tissue-targeted with receptor-mediated gene delivery. After a unique receptor protein in the tissue of interest has been identified, a natural ligand or an antibody to the receptor can be conjugated to polylysine or some other coupling agent to deliver DNA.59
Transgene Expression Is Unregulated
Most vascular gene transfer approaches have used viral promoters
to drive constitutive gene expression. However, vascular gene therapy
may require regulating gene expression over time to titrate therapeutic
effects. The use of inducible promoters would allow the administration
of exogenous agents that could drive transgene expression. Several
types of regulatable vectors have been developed. An example is a
system based on the Escherichia coli tetracycline-responsive
promoter (tet).60 A `gene switch'
is thereby constructed that allows the transgene to be specifically
turned on/off in response to tetracycline.
An intriguing concept is the regulation of transgene expression by endogenous stimuli such as inflammation and hypoxia. Placing a transgene under the control of a cytokine-inducible promoter may allow production of the recombinant protein only in the setting of an inflammatory response. The intensity and duration of the inflammatory response would then drive transgene expression.61 Similarly, hypoxia, a potent modulator of gene expression,62 63 could be used for regulating transgene expression. Modulation caused by hypoxia results from the interaction of a transcriptional complex called "hypoxia-inducible factor-1" (HIF-1), with its DNA recognition site, the hypoxia response element (HRE).64 65 This HIF-1-HRE–dependent system of inducible gene expression is present in all mammalian cells that have been tested66 and may be useful in directing gene expression in ischemic myocardium or skeletal muscle.63
Delivery of Vectors to the Vasculature
Delivery of vectors for gene transfer into specific
arterial beds poses significant technical challenges unique
to each anatomical site. Ideally, a system to deliver vectors to a
vascular bed should be catheter based, permit distal perfusion of the
target organ while the vector is being delivered, and allow subsequent
retrieval of the vector to avoid the potential dangers of systemic
dissemination. Vector delivery may have to be made to an entire
arterial bed (to ameliorate conditions like
atherosclerosis and transplant vasculopathy) or it may
be site- specific (to modulate localized arterial lesions).
Transduction of an entire vascular bed would require advanced catheter
techniques or it could be achieved at the time of arterial
bypass surgery or organ transplantation.67 For
site-specific gene delivery, the branching nature of the
arterial bed and the potential for ischemic damage
from prolonged balloon inflation pose significant challenges. In both
situations, unknown transfection efficiency with advanced
atheromatous disease and the possibility of systemic
dissemination of the vector need to be considered.
Initially, in vivo gene transfer to the vasculature involved infusion of the vector into a segment of an artery isolated by sealing it at 2 locations with a double-balloon catheter.21 Next, efforts were made to develop perfusion catheters that permitted blood flow through an artery while delivering vectors. Such catheters included a modification of the double-balloon catheter,68 the channel balloon (which has an array of channels with pores surrounding an inner balloon),69 and the Dispatch catheter (which isolates a segment of an artery, using a thin polymer membrane on a spiral balloon).70 Another method of gene delivery takes advantage of the hydrophilic polymer (hydrogel) coating present on some angioplasty catheters. A vector solution contained in the polymer is delivered to the vessel wall with balloon inflation.71 Recently, a "needle injection" catheter has been developed that allows direct gene transfer to the adventitia or the media.35 This is achieved by multiple small needles, which are arranged linearly along the surface of the balloon, that penetrate the vessel wall as the balloon is inflated. Finally, stents need to be considered as potential vehicles for gene transfer because of their increasing use in vascular interventions. The feasibility of coating stents with genetically engineered ECs has been demonstrated.15 16 A biopolymer coating such as fibrin polymer can be used to cover the metal scaffolding of a stent and as a vehicle to deliver transduced cells or vectors to the vessel wall.72 Similarly, stents made entirely of a bioresorbable polymer may prove to be useful adjunctive devices for vascular gene transfer.73 Successful gene transfer and expression have been demonstrated after implantation of polymeric stents impregnated with an adenoviral vector encoding ß-galactosidase in rabbit carotid arteries.74
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Part II: Therapeutic Potential |
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TopIntroductionPart I: Basic PrinciplesPart II: Therapeutic PotentialReferences |
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Part II is an overview of the potential use of gene transfer in the treatment of vascular diseases, including atherosclerosis, arterial restenosis, vein graft disease, systemic and pulmonary hypertension, and cerebrovascular disease. Most of the discussion is based on studies performed in animal arteries that do not have the same lesions as diseased arteries of humans. At best, these studies are "proof-of-principle" studies, setting the stage for further testing in models more representative of human disease or in humans themselves.
Atherosclerotic Vascular Disease
Atherosclerosis is a complex disorder. Vascular
gene transfer strategies for atherosclerosis may be
directed at atherogenesis itself or at the important sequelae of
atherosclerosis, such as thrombosis and
ischemia.
Antiatherogenic Strategies
Early atherogenesis is characterized by
endothelial dysfunction that manifests as impaired
endothelium-dependent relaxations and increased
adhesiveness to circulating white blood cells. Decreased
bioavailability of NO, from either decreased production or due
to scavenging by free oxygen radicals, seems to be a key factor in
endothelial dysfunction.75 NO has several
antiatherogenic properties.76 In addition to its role as
an endogenous vasodilator, it inhibits SMC growth and
migration, nuclear factor kappa B (NF-
B) activity,77
and the expression of proinflammatory molecules such as vascular cell
adhesion molecule-1 and monocyte chemoattractant
protein-1.78 79 Endothelial function may
be enhanced and atherogenesis retarded by increasing local NO
production through overexpression of eNOS.24
However, increased NO production may not necessarily be
beneficial in advanced atherosclerosis.80
Indeed, there is potential for increased production of damaging
free radicals such as superoxide and peroxynitrite, with resulting
vascular damage.81 82 Coexpression of transgenes that
encode for antioxidant proteins might be useful in such a
setting.83 84 Other antiatherogenic proteins that are
candidates for gene therapy for atherosclerosis include
apo A-I, the principal apolipoprotein of high-density lipoprotein,
which promotes reverse cholesterol transport in
atherosclerotic lesions,85 86 and apo E, the ligand
involved in clearance of lipoprotein particles from the
circulation.87 88
Thrombosis
Most acute ischemic syndromes result from thrombosis
subsequent to rupture of an atheromatous plaque.
Thrombin, a central molecule in blood coagulation, may be inhibited by
expression of recombinant hirudin.89 Other antithrombotic
approaches include overexpression of cyclooxygenase
to augment prostacyclin synthesis90 and overexpression of
tissue plasminogen activator to enhance
fibrinolysis.91 The delay between
administration of vector and the onset of antithrombotic effects means
that such a strategy is unlikely to be effective in acute settings.
Gene transfer to "stabilize" the "vulnerable" atherosclerotic
plaque may prevent plaque rupture and subsequent thrombosis. Possible
strategies include overexpressing TIMPs and blocking the action of
proinflammatory molecules such as the transcription factor
NF-B.92 Further progress in this area awaits the
development of methods to identify vulnerable plaques and the
establishment of animal models of plaque rupture.
Angiogenic Gene Therapy
This is an option for severe occlusive atherosclerotic
arterial disease refractory to medical therapy and not
amenable to mechanical revascularization. Direct
administration of angiogenic proteins has several disadvantages,
including cost, need for repeated or prolonged administration of
protein, and systemic effects. Gene transfer of angiogenic factors is a
viable alternative therapy. Vascular endothelial growth
factor (VEGF) is an EC mitogen in vitro and an angiogenic growth factor
in vivo.93 In addition, it may have qualitative effects on
endothelial function,94 including
stimulation of NO production.95 In the rabbit
ischemic hindlimb model, augmented collateral perfusion
occurred after delivery of plasmid DNA encoding VEGF.96 A
trial with human subjects is studying the efficacy of such a method for
patients with critical peripheral arterial
disease.97 A plasmid encoding VEGF is delivered to the
vasculature proximal to the site of narrowing with use of a hydrogel
balloon catheter. Preliminary results seem encouraging, at least in 1
patient.98 An alternate strategy is intramuscular delivery
of VEGF gene in patients with severe diffuse peripheral
arterial disease in whom conventional treatment has failed
and intravascular catheter-based gene delivery is not possible.
Skeletal muscle is able to take up and express naked plasmid DNA. With
this method of delivery, augmented collateral development and tissue
perfusion were demonstrated in the rabbit model of hindlimb
ischemia.99 Results using such a strategy in 10
patients with severe peripheral arterial
disease (7 with nonhealing ulcers/digit gangrene, 3 with rest pain)
have been published recently.100 Clinical status was
improved in 8 patients, unchanged in 1, and worse in 1 after 2 to 6
months of follow-up. An increase in the ankle-brachial index, improved
flow by magnetic resonance imaging, and angiographic evidence of
angiogenesis were noted in several patients.
Successful angiogenic gene therapy for myocardial ischemia has been demonstrated in a porcine model of ameroid constrictor–induced coronary occlusion.101 Intracoronary delivery of a recombinant adenovirus expressing human fibroblast growth factor (FGF)-5 resulted in marked improvement in myocardial function, blood flow, and increased capillary density. Clinical trials with human subjects involving the adenoviral-mediated expression of FGF-4 to stimulate angiogenesis in ischemic myocardium are under way.
Arterial Restenosis
To develop effective molecular therapies for restenosis,
it is important to understand its biology. During the past several
years, our understanding of the pathogenesis of restenosis has
changed considerably.102 The concept that luminal loss in
restenosis results simply from an increase in intimal mass is
no longer valid. Although SMC proliferation is possibly the main
mechanism for restenosis after stenting, factors such as
thrombosis, platelet activation, vasoconstriction, leukocyte
adhesion, matrix elaboration, and remodeling are important pathogenetic
mechanisms in other settings.103 Vascular remodeling may
be a major determinant of whether restenosis will occur, and
its importance has been highlighted in several animal
models104 105 and in humans.29 Therefore,
gene therapy approaches to limit restenosis need to be targeted
not only at cell proliferation but also at other pathogenetic factors.
The various gene transfer strategies that have been used experimentally
to limit neointimal formation and restenosis are
discussed below.
Antiproliferative Strategies
Gene therapy may be used to decrease cell number in the vascular
wall by inducing either cytotoxicity or cytostasis (Table 4). With mathematical modeling of cell
proliferation, a significant and clinically relevant inhibition of
neointima formation is predicted at cell transduction rates
as low as 10%.116 However, this modeling does not take
into account the contribution of SMC migration to neointima
formation and the "bystander" phenomenon thought to occur after
HSV-tk gene transfer. The question whether gene transfer for
antiproliferative effects is better than drugs such as paclitaxel
(Taxol) or plicamycin (Mithramycin) or radiation needs to be
considered. With systemically administered antiproliferative
medications, the potential for serious side effects is considerable.
Similarly, with the use of arterial radiation, the
possibility of long-term vascular damage needs to be considered.
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Inhibition of SMC Migration
SMC migration and matrix elaboration are important in the
pathogenesis of restenosis.117 Migration of SMCs
is mediated partly through the action of
metalloproteinases.118 Local overexpression of a TIMP-1 in
injured carotid arteries of rats produced a significant decrease in
neointimal hyperplasia.5 Overexpression of
proteins such as eNOS or natriuretic peptides, which
increase vascular wall cGMP, may also inhibit SMC
migration.119 120
Inhibition of Thrombosis
Thrombin is important in the vascular response to injury and is an
attractive target for pharmacological inhibition in
vivo.121 Adenoviral-mediated gene transfer of recombinant
hirudin, a specific inhibitor of thrombin, decreased
neointima formation in the rat carotid artery injury
model.89 In a porcine carotid artery injury model,
adenoviral-mediated transfer of the cyclooxygenase
gene augmented prostacyclin synthesis and was associated with
inhibition of thrombosis.90 Neointimal
formation was not studied in this model, but such an approach may be
useful in limiting restenosis.
Acceleration of Endothelialization
Studies in the rat carotid artery model of arterial
balloon injury have established an inverse relationship between
endothelial integrity and SMC
proliferation.122 Direct application of VEGF to the
denuded surface of the rat carotid artery accelerates
endothelialization and decreases intimal
proliferation.123 Catheter-mediated gene transfer of a
plasmid encoding VEGF to the balloon-injured site of a rabbit femoral
artery led to accelerated re-endothelialization at
local and remote sites, leading to inhibition of neointimal
thickening, reduction of thrombogenicity, and restoration of
endothelium-dependent relaxations.124 A
trial with human subjects is under way to test this strategy in
peripheral artery angioplasty.125 However,
several aspects of VEGF biology need to be considered as trials with
human subjects begin. Upregulation of VEGF has been demonstrated in
medial SMCs after balloon injury,126 which raises the
question of the added effectiveness of recombinant VEGF in this
situation. VEGF may have mitogenic effects on
SMC127 and thus could exacerbate neointimal
formation.128 Also, VEGF may induce plaque
neovascularization in atherosclerotic arteries, with possible
deleterious effects.
Pleiotropic Effector Molecules
Because of the more complex vascular lesions in humans, with
involvement of several pathogenetic mechanisms that include
inflammation and platelet-fibrin thrombus
deposition,117 129 a "cocktail approach" may be
necessary to affect multiple mechanisms. Thus, a mixture of different
adenoviral vectors or an adenoviral gene that encodes 2 or more gene
products may prove successful. Alternatively, agents with
pleiotropic actions are more likely to be effective in limiting
neointimal formation than those that act on a single
pathogenetic mechanism.130 Two such molecules are NO and
C-type natriuretic peptide (CNP), both of which increase
cGMP in the arterial wall.
NO is important in the regulation of vascular tone and the prevention of thrombosis. NO donors inhibit SMC proliferation,131 migration,120 and matrix production,132 133 and platelet134 and monocyte135 adhesion. These factors are important in determining neointimal formation, particularly after balloon angioplasty or stenting. Increasing local NO production therefore may uniquely inhibit formation of neointima in vascular disease states. Also, because NO has a role in vascular remodeling,136 increased NO generation may favor positive remodeling in balloon-injured arteries.6 Thus, localized eNOS delivery conceptually has several advantages over other forms of gene therapy to limit restenosis. eNOS gene transfer to the injured carotid artery of rats led to a reduction in neointima.137 Retroviral-mediated gene transfer of the inducible isoform of NOS has also been demonstrated to inhibit myointimal hyperplasia in an ex vivo organ culture model of arterial balloon injury.138 However, as mentioned above, the effects of NO may depend on the redox state of the vessel wall. In states of excessive oxidant stress such as atherosclerosis or vascular injury, increased NO generation may lead to formation of damaging free radical oxidant species such as peroxynitrite.
CNP is a member of the natriuretic peptide family with vasorelaxing, antimitogenic, and anti–smooth muscle migratory properties.139 Most of the actions are mediated by the cGMP pathway. Adenoviral-mediated gene transfer of CNP caused a marked decrease of neointimal formation in the rat carotid artery injury model.140 Because the antiproliferative effects of the vector on SMC in vitro were modest, it is likely that neointimal formation in vivo is limited by several different mechanisms.140
Vein Graft Disease
Occlusion of vein grafts is a major limitation to the long-term
treatment of occlusive arterial disease. Mann and
coworkers141 demonstrated that inhibition of cell
proliferation using antisense technology prevents accelerated
atherosclerosis and neointimal formation in
jugular vein bypass grafts of cholesterol-fed rabbits. The
treated grafts develop medial hypertrophy as an adaptation
to increased hemodynamic stress, are resistant
to diet-induced atherosclerosis, and have preserved
endothelial function.141 142 On the basis
of these findings, a multicenter trial of intraoperative ex vivo
delivery of E2F decoy oligonucleotides to vein grafts
in humans to decrease the incidence of graft disease and failures has
begun.20 E2F is a transcription factor that mediates
coordinated induction of cell cycle regulatory genes.143
An alternate strategy of enhancing endothelial function
and antithrombotic properties of vein grafts is to increase NO
production by expression of recombinant eNOS.
Adenoviral-mediated eNOS gene transfer ex vivo to saphenous vein grafts
in humans resulted in enhanced relaxations to calcium ionophore and
increased nitrite production by treated vein
grafts.19 Such a strategy may decrease the incidence of
acute graft occlusion and perhaps allow favorable remodeling.
Systemic and Pulmonary Hypertension
Although hypertension is a polygenic disease, certain key
mediators may have significant roles that could be modified by gene
transfer. Gene therapy may allow chronic hypertension control,
obviating daily lifelong drug therapy. Direct intravenous
delivery of plasmid DNA encoding vasodilator proteins has been tested
in the spontaneously hypertensive rat (SHR). Plasmid DNA encoding human
tissue kallikrein decreased blood pressure for 6 weeks, with a peak
decrease of 46 mm Hg.144 Similar delivery of the
atrial natriuretic peptide gene led to reduction in blood
pressure after 1 week; the effect lasted up to 7 weeks and was
accompanied by diuresis and natriuresis.145 A
similar prolonged decrease in blood pressure was obtained by
intravenous delivery of a plasmid encoding
eNOS.146 A novel method of increasing vascular NO
production was adopted by Cuevas and coworkers,147
who observed decreased endothelial basic FGF content in
the endothelium of the SHR. Intravenous
delivery of a plasmid encoding basic FGF led to increased eNOS
expression in the endothelium and attenuated responses
to vasoconstrictors.
Primary pulmonary hypertension is a disorder that can be rapidly fatal and for which there is no effective therapy. The expression of eNOS in the pulmonary vasculature of patients with primary pulmonary hypertension is markedly diminished.148 A novel therapeutic approach to this condition may be to increase eNOS activity in the lungs of patients by gene transfer techniques. Adenoviral-mediated expression of human recombinant eNOS in rat lungs has been shown to attenuate hypoxic vasoconstriction.36 Recombinant adenovirus was delivered by aerosolization and localized in part to the adventitia of the pulmonary arteries.
Cerebrovascular Disease
Vasospasm is a major cause of mortality and morbidity after
subarachnoid hemorrhage. Expression of transgenes such
as eNOS in the cerebral vessels may result in vasodilatation and
ameliorate vasospasm in such a situation.37 149 In a
canine model of subarachnoid hemorrhage, intracisternal
injection of recombinant adenovirus encoding a reporter gene resulted
in gene transfer to the cerebral blood vessels and overlying meninges
even in the presence of cisternal blood.150 Although
adenoviral vectors allow only transient expression of transgenes, this
may be advantageous in the setting of subarachnoid
hemorrhage because spasm typically occurs within 7 to 10 days
after the hemorrhage, at a time when transgene expression
should still be present.
Summary
Several important insights have been gained after nearly a decade
of investigation in vascular gene transfer. In animal models, vascular
gene transfer has been used successfully to alter
neointimal formation and vasomotor reactivity and to
stimulate angiogenesis. However, the shortcomings of such an approach
for treating vascular disorders in humans have become apparent. Effects
of gene transfer in animal models may not be applicable to diseased
human arteries, and more representative animal models
need to be established. Currently available vectors have several
deficiencies such as transient expression, generation of an
immune-inflammatory response, lack of tissue specificity, and
unregulated expression. Better percutaneous
catheter-based delivery systems need to be developed. Safety and
cost-effectiveness issues also need to be considered.
Despite these imperfections, gene therapy for vascular diseases in humans has already begun. This has occurred because of certain unique scenarios rather than the maturation of the field. Gene therapy trials with human subjects to stimulate angiogenesis in ischemic skeletal or cardiac muscle are an example. The first trial involved plasmid-mediated VEGF delivery to stimulate angiogenesis in critically ischemic limbs.97 Factors such as lack of effective alternative therapy in patients with severe symptomatic peripheral arterial disease, upregulation of VEGF receptors in the setting of ischemia (increasing the chances of obtaining biological effects despite the low efficiency of gene transfer with plasmid-mediated delivery), and presumed endothelial specificity of VEGF receptors led to the approval of a trial with human subjects. A separate arm of this protocol tested the direct administration of naked VEGF DNA into skeletal muscle.100 This was feasible because skeletal muscle takes up and expresses naked DNA relatively efficiently. Trials of gene transfer are underway to stimulate angiogenesis in chronic severe coronary artery disease for which conventional medical and surgical therapeutic options were exhausted. The stimulus for such trials is the lack of effective therapeutic options in the setting of disabling symptoms, rather than a refinement of gene transfer techniques. More such trials are likely to get under way in the setting of severe vascular disease. In such scenarios, the risk of therapeutic uncertainty and vector imperfections (present with the current first- or second-generation adenoviral vectors) may have to be accepted.
Better understanding of vascular pathobiology, continuing basic science research aimed at refining vectors and delivery systems, and insights from the early trials with human subjects are critical for a broader use of vascular gene transfer. With such developments and close cooperation between clinicians and basic scientists, vascular gene transfer is likely to emerge as an important therapeutic modality for vascular disorders.
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Acknowledgments |
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The work was supported by the Mayo Foundation Clinician-Investigator Training Program (I.J.K.), by the Minnesota affiliate of the American Heart Association (I.J.K.), and by the Bruce and Ruth Rapapport Program in Vascular Biology (R.D.S).
Received December 12, 1997; accepted July 10, 1998.
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References |
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