Cytostatic gene therapy for occlusive vascular disease
José M. González and Vicente Andrés
Laboratory of Vascular Biology, Department of Molecular and Cellular Pathology and Therapy,
Instituto de Biomedicina de Valencia (IBV-CSIC), Jaume Roig 11,Valencia 46010, Spain.
Send correspondence to:
Vicente Andrés (Tel: +34-96-3391752; FAX: +34-96-3391751; E-mail: vandres@ibv.csic.es)
KEY WORDS: atherosclerosis, restenosis, bypass graft failure, cell cycle, gene therapy
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1. Introduction
2. Antiproliferative gene therapy in animal models of occlusive vascular disease
2.1. Cell cycle regulatory genes
Inactivation of positive cell cycle regulatory genes
Overexpression of growth suppressors
2.2. Growth factors
2.3. Transcription factors involved in cell cycle control
2.4. Miscellaneous
3. Antiproliferative gene therapy clinical trials for human occlusive vascular disease
3.1. E2F
3.2. c-myc
3.3. VEGF
4. Expert opinion
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Abstract
The formation of occlusive vascular lesions during the course of atherosclerosis, in-stent
restenosis, transplant vasculopathy, and vessel graft failure is a chronic inflammatory process
characterized by excessive cellular proliferation within the injured artery wall. Therefore,
candidate targets for the treatment of vasculoproliferative disease include cell cycle regulatory
factors, such as cyclin-dependent kinases (CDKs), cyclins, CDK inhibitory proteins (CKIs),
tumor suppressors, growth factors and their receptors, and transcription factors involved in cell
cycle control. Although several genetically-modified mouse models have conclusively
demonstrated that increased cell proliferation aggravates atheroma development, the potential
benefit of cytostatic strategies for the treatment of atherosclerosis in clinic is doubtful because
human atherosclerosis is often diagnosed at advanced stages when neointimal proliferation
appears low or absent. In contrast, restenosis and graft atherosclerosis appear amenable for
cytostatic strategies because neointimal lesions typically develops over a short period of time
after revascularization (e. g., 2-12 months) and is localized at the site of the intervention.
Vascular interventions, both endovascular and open surgical, allow minimally invasive, easily
monitored gene delivery. In this review, we will discuss preclinical studies and clinical trials
utilizing cytostatic gene therapy for occlusive vascular disease.
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1. Introduction
Atherosclerosis and associated cardiovascular disease (e.g., myocardial infarction and
stroke) are the main cause of mortality and morbidity in industrialized countries, and their
incidence in developing countries is increasing at an alarming rate. Atherosclerosis is a chronic
inflammatory disease of middle-sized and large-caliber arteries that normally progresses over
several decades and may remain silent until fatal manifestations occur at advanced disease
stages. Endothelial damage caused by several risk factors, such as hyperlipemia, hypertension,
diabetes, and smoking, is considered the first manifestation of occlusive vascular disease (e. g.,
atherosclerosis, in-stent restenosis, transplant vasculopathy, and vessel graft failure) [1, 2]. Both
acellular (e.g., deposition of lipids and extracellular matrix components) and cellular components
are involved in the growth of neointimal lesions. Accumulation of neointimal cells occurs via
both transendothelial migration of circulating leukocytes and migration of vascular smooth
muscle cells (VSMCs) from the tunica media towards the atheroma, and from excessive
proliferation of nointimal monocyte/macrophages and VSMCs. Rupture or erosion of advanced
atherosclerotic plaques can lead to thrombus formation and acute ischemic events (e.g.,
myocardial infarction and stroke).
Percutaneous transluminal angioplasty (PTA) and vessel by-pass grafting are widely used
interventions for revascularization of obstructed vessels. Although both procedures have a high
rate of initial success, their long-term efficacy is often limited by luminal narrowing, typically
within 3-12 months after intervention. Among other factors, abundant neointimal cell
proliferation contributes to restenosis after angioplasty and vessel graft failure [3-5].
Neointimal thickening is a complex process initiated and sustained by growth factors and
their receptors, signal transduction pathways and transcription factors that ultimately control de
activity of CDK/cyclins (see below). In the following sections, we will discuss gene therapy
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strategies designed to prevent cellular proliferation for the treatment of occlusive vascular
disease, both in experimental animals and in the clinical setting.
2. Antiproliferative gene therapy in animal models of occlusive vascular disease
2.1. Cell cycle regulatory genes
Cell cycle progression in higher eukaryotes is positively regulated by several
holoenzymes composed of a catalytic and a regulatory subunit, named CDK and cyclin,
respectively [6] (Fig. 1). Activation of CDK/cyclins by mitogenic stimuli causes the
hyperphosphorylation of the retinoblastoma protein (pRb) and the related pocket proteins p107
and p130 from mid G1 to mitosis. The complex interaction among E2F transcription factors and
individual pocket proteins determines whether E2F proteins function as transcriptional activators
or repressors [7]. Proteins of the CKI family promote growth arrest by inhibiting CDK activity
via interaction with CDK/cyclin complexes. CKIs of the Cip/Kip family (p21Cip1, p27Kip1 and
p57Kip2) bind to and inhibit a wide spectrum of CDK/cyclin holoenzymes, while members of the
Ink4 family (p15Ink4b, p16Ink4a, p18Ink4c, and p19Ink4d) are specific for cyclin D-associated CDKs.
Mitogenic and antimitogenic stimuli affect the rate of synthesis and degradation of CKIs, as well
as their redistribution among different CDK/cyclin pairs [6].
Inactivation of positive cell cycle regulatory genes
Arterial cell proliferation in response to balloon angioplasty in the rat carotid artery is
associated with a temporally and spatially coordinated expression of CDKs and cyclins. [5] [8].
Importantly, augmented expression of these factors correlated with CDK2 and CDC2 activation,
demonstrating the assembly of functional CDK/cyclin holoenzymes within the injured arterial
5
wall. CDK2 and cyclin E expression has been detected in human VSMCs within atherosclerotic
and restenotic tissue [9], suggesting that increased expression (and possibly activation) of
positive regulators of cell cycle progression is a characteristic of vasculoproliferative disease in
humans.
Neointimal thickening in animal models of balloon angioplasty is limited by antisense
oligodeoxynucleotide (ODN) strategies targeting CDKs and cyclins, including cdk2 [10] [11],
cdc2 [10] [11], and cyclin B1 [11]. Cotransfection of antisense ODN against cdc2 and cyclin B1
was more effective at reducing neointimal thickening than blockade of either gene target alone
[11]. Likewise, combined inactivation of cdc2 and proliferating cell nuclear antigen (pcna) by a
single intraluminal delivery of antisense ODNs resulted in sustained inhibition of neointima
formation in the rat carotid artery balloon-injury model [12]. However, this approach was
ineffective in balloon-injured porcine coronary arteries [13]. The use of hammerhead ribozyme
to pcna alone has been successful in reducing in-stent restenosis in a porcine coronary model
[14]. Inactivation of cyclin G1 gene expression by retrovirus-mediated antisense gene transfer
inhibited VSMC proliferation and neointima formation after balloon angioplasty [15]. Moreover,
ODN against cdk2 [16], and a combination of antisense ODN against pcna and cdc2 [17],
attenuated vessel graft failure in experimental animals.
Immusol Inc. has described methods of producing ribozymes especially targeted to cyclin
B1, cdc2, and pcna to inhibit VSMC proliferation in vascular tissue, as well as ribozyme delivery
systems for anti-restenosis gene therapy [301]. Another method based on siRNA inhibition of
CDK4 activity has been claimed for preventing or treating cancer [302], which could also find
application for treating vascular proliferative disease.
Overexpression of growth suppressors
CKIs
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The efficacy of CKIs as cell cycle suppressors has been widely documented in a variety of
normal and tumor cells in vitro. Evidence suggesting that the CKIs p21Cip1 and p27Kip1 play
important roles in cardiovascular pathophysiology includes the following: 1) p21Cip1 and p27Kip1
expression is upregulated at late time points after balloon angioplasty in rat and porcine arteries
coinciding with the reestablishment of the quiescent phenotype after the initial proliferative
response [18] [19]; 2) p27Kip1 may function as a molecular switch that regulates the phenotypic
response of VSMCs to both hyperplastic and hypertrophic stimuli [20, 21]; 3) p27Kip1 is a
negative regulator of endothelial cell (EC) proliferation and migration in vitro [22], and
adenovirus-mediated overexpression of p27Kip1 inhibited angiogenesis in vivo [23]; 4) p21Cip1 and
p27Kip1 may contribute to integrin-mediated control of VSMC proliferation [24]; 5) p27Kip1 limits
cardiomyocyte proliferation during early postnatal development and after injury in adult mice
[25] [26]; 5) expression of p27Kip1 and p21Cip1 is more frequent within regions of human coronary
atheromas not undergoing proliferation [19]; 6) intrinsic differences in the regulation of p27Kip1
may contribute to establishing regional variability in atherogenicity via distinct regulation of
VSMC proliferation and migration [27].
Analysis of genetically-modified mice further supports the notion that CKIs are key
regulators of neointimal lesion development. Genetic p27Kip1 ablation, either global or selectively
in hematopoietic precursors, accelerates arterial cell proliferation and aggravates atherosclerosis
in apolipoprotein E (apoE)-deficient mice [28] [29]. Surprisingly, both global and hematopoietic
cell-specific disruption of p21Cip1 in apoE-null mice protects from atherosclerosis, possibly due to
effects of this CKI on macrophage function and inflammatory responses that appear independent
of its cell cycle regulatory function [30].
Global genetic inactivation of either p16Ink4a [31] or p21Cip1 [32] exagerates neointimal
thickening induced in the mouse by mechanical vessel denudation. Regarding the effect of
p27Kip1 inactivation on neointimal lesion formation after mechanical arterial injury, Roque et al.
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found similar lesion size in wild-type and p27Kip1-null mice [33]; however, Boehm et al. reported
a marked increase in mechanically-induced neointimal tickening in p27Kip1-null mice [34].
Intraluminal delivery of replication-defective adenoviral vectors encoding p21Cip1 and
p27Kip1 reduced neointimal thickening in rat, porcine and murine models of balloon angioplasty
[18, 35-39]. Neointimal lesion formation in a rabbit model of vein grafting was also attenuated by
ectopic overexpression of p21Cip1 [40], and adenovirus-mediated transfer of a p27 Kip1-p16
Ink4a
fusion gene inhibited neointimal hyperplasia in balloon-injured porcine coronary [41] and
cholesterol-fed rabbit carotid arteries [42].
Methods of preparation and use of recombinant adenoviral vectors capable of expressing
human p21Cip1, p27Kip1, p16Ink4a and other growth suppressors have been described for inducing
growth arrest of proliferating cells, as well as methods for the eradication of cancer and diseased
cells [303]. Likewise, methods of inhibiting cell proliferation using purified p18 Ink4c or p19Ink4d
proteins, and methods of gene therapy using nucleic acids that encode these genes have been
described [304, 305]. The University of Texas System has claimed the use of non-viral and viral
expression vectors encoding for mutant p21Cip1 proteins as a therapy for the treatment of
proliferative cell disorders, including cancer, restenosis, neurogenerative disease and
angiogenesis-related conditions [306]. The mutations consist of Thr145Ala or Thr145Asp
substitutions, which result in nuclear retention or cytoplasmic translocation of p21Cip1,
respectively, which result in preferential suppression of cell growth (p21Cip1 Thr145Ala), or
enhanced cell survival (p21Cip1 Thr145Asp).
Reagents and methods for identifying genes whose expression is modulated by induction
of CKI gene expression have been presented [307]. This invention also provides reagents and
methods for identifying compounds that inhibit or augment the effects of CKIs, such as p21Cip1
and p16Ink4a, on cellular gene expression, as a first step in rational drug design for preventing
cellular senescence, carcinogenesis and age-related diseases (such as atherosclerosis and related
8
disease), or for increasing the efficacy of anticancer therapies.
A method for treating vascular proliferative diseases by administering in vivo a gene
encoding p27Kip1 has been patented [308]. Proteasome degradation of p27Kip1 is thought to play a
major role in the regulation of p27Kip1 expression. Kyushu University has patented the nucleic
acid and amino acid sequence of a new p27Kip1 molecular variant exhibiting resistance to
proteasome degradation, as well as expression vectors encoding this derivative of p27Kip1 for
gene therapy applications targeting cell propagating lesions, such as tumours and atherosclerotic
plaques [309].
p53
The tumor suppressor p53 displays both antiproliferative and proapoptotic actions (Fig.
2). These effects result from transcriptional activation of antiproliferative and proapoptotic genes
(e. g., p21Cip1 and Bax, respectively), transcriptional repression of proproliferative and
antiapoptotic genes (e. g., IGF-II and bcl-2, respectively), and direct protein-protein interactions
(e. g., with helicases and caspases). Antisense p53 ODN transfection [43, 44] or p53 gene transfer
[45] increases or decreases VSMC proliferation, respectively, and VSMCs isolated from p53deficient mice exhibit more proliferative and migratory activity than their wild-type counterparts
[46].
p53 is overexpressed but not mutated in human atherosclerotic tissue [47]. Because lack
of proliferation markers in vascular cells within advanced human atherosclerotic lesions
coincided with p53 and p21Cip1 coexpression, Ihling et al. suggested that p53-dependent
transcriptional activation of p21Cip1 might protect against excessive vascular cell growth [48].
However, it is noteworthy that p21Cip1 expression aggravates atherosclerosis in apoE-null mice
[49]. Both global and hematopoietic cell-specific p53 genetic ablation results in increased
atherosclerosis in several murine models, including apoE-null, apoE*3-Leiden transgenic, and
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LDL receptor-null mice, although the relative contribution of cellular proliferation and apoptosis
in these animal models remains unclear [50-53]. Genetic disruption of p53 in the mouse also
accelerated neointimal lesion induced by vein grafting [46] and external vascular cuff placement
[54].
Both animal and human studies suggest that p53 plays an important role in the
pathogenesis of restenosis. Intraluminal transfection of antisense p53 ODN into rat carotid artery
or p53 genetic inactivation in mice accelerated neointimal hyperplasia after vascular injury [44,
55]. It has been suggested that increased VSMC proliferation and migration via inactivation of
p53 in response to human cytomegalovirus infection contributes to the development of
atherosclerosis and restenosis [56-59]. Compared to primary cultures of human VSMCs isolated
from normal vessels, VSMCs from restenosis or in-stent stenosis sites exhibit normal or
enhanced responses to p53 [60]. Moreover, p53 gene transfer effectively inhibited neointimal
hyperplasia after experimental angioplasty [45] and in organ cultures of human saphenous vein
[61].
Introgen Therapeutics Inc has claimed a method of inhibiting the growth of human
papillomavirus-transformed keratinocytes and to prevent or suppress papillomavirus-mediated
cell transformation by topically administering a p53 expression cassette carried in either a viral or
non-viral vector, which could be administered with a secondary anti-hyperplastic therapy [310].
pRb
The complex interplay between pRb and transcription factors of the E2F family plays a
critical role in the control of cell growth [7] (Fig. 1). In quiescent cells, E2F-dependent
transactivation of genes required for cell cycle progression is prevented, at least in part, by the
accumulation
of
hypophosphorylated
pRb.
In
contrast,
mitogen-induced
pRb
hyperphosphorylation leads to E2F activation and cell proliferation. Inactivation of pRb by
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antisense ODN resulted in the induction of the proapoptotic factors bax and p53, increased
number of apoptotic cells and a higher rate of DNA synthesis in human VSMCs [43]. Growth
arrest of cultured VSMCs and attenuation of neointima formation after balloon angioplasty can
be achieved by adenovirus-mediated transfer of several forms of pRb, including full-length
constitutively active (nonphosphorylatable) and phosphorylation-competent pRb, and truncated
versions of pRb [62, 63]. Similarly, adenoviral transfer of the pRb related protein RB2/p130
inhibited VSMC proliferation in vitro and prevented neointimal hyperplasia after experimental
angioplasty [64]. Adenoviral transfer of a constitutively active mutant pRb protein also inhibited
neointima formation in a human explant model of vein graft disease [65]. Likewise, targeting
overexpression of an anti-proliferative pRb/E2F hybrid transgene to VSMCs using the human
smooth muscle α-actin promoter suppresses cell proliferation and neointima formation [66].
2.2. Growth factors
Growth factors that have been implicated in the regulation of VSMC proliferation and
migration include platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), tumor
necrosis factor- (TNF- epidermal growth factor (EGF), insulin-like growth factor-1 (IGF1), heparin-binding epidermal growth factor-like growth factor, interleukin-1 and transforming
growth factor-TGF. Expression of these factors is increased during atherogenesis [67] and
restenosis after angioplasty [68].
TGF-
TGF1 regulates extracellular matrix synthesis, cell chemotaxis, and proliferation in
VSMCs, ECs and fibroblasts. Adenovirus-mediated antisense TGF-1 treatment reduces vein
graft intimal hyperplasia [69]. Likewise, ribozymes targeted to human TGF-1 significantly
inhibited angiotensin II-stimulated human VSMCs growth [70] and neointimal formation after
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balloon injury in the rat carotid artery [71] [72]. However, adenovirus-dependent gene transfer of
TGF-1 does not affect luminal loss after porcine coronary angioplasty, while adenovirusmediated TGF-3 delivery increased adventitial collagen content and the external elastic lamina
area, and reduced luminal loss in this model [73].
IGF-1
IGF-1 has been implicated in the control of VSMC proliferation and locomotion [74].
Adenovirus-dependent transfer of a dominant negative truncated mutant of IGF-1 receptor in
cultured VSMCs attenuated serum- or IGF-1-dependent phosphorylation of Akt and ERK1/2,
inhibited migration and proliferation and increased apoptosis [74]. This strategy also reduced
neointima formation after balloon angioplasty in the rat carotid artery [74].
EGF
The EGFR family consists of four receptor tyrosine kinases, EGFR (ERBB1), ERBB2
(HER2), ERBB3 (HER3) and ERBB4 (HER4), which specifically interact with approximately a
dozen of EGF-like growth factors [75]. Genentech Inc has patented a method based on the
administration of an antagonist of a native ErbB4 receptor for preventing excessive VSMC
proliferation or migration and for the treatment of stenosis or restenosis [316].
FGF
Recombinant FGF-1 promoted neointimal hyperplasia [76]. Conversely, neutralizing
antibodies directed against basic FGF (bFGF or FGF-2) inhibited neointimal VSMC accumulation
after angioplasty [77], and gene transfer of a soluble FGF receptor 1 molecule capable of
sequestering circulating FGF-1 and FGF-2 reduced the development of accelerated graft
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arteriosclerosis in a rat aortic transplant model [78]. Inhibition of human FGF receptor 2
expression by antisense ODN has been claimed for treating hyperproliferative disorders [317].
PDGF
PDGF promotes proliferation of a wide range of cell types, including fibroblasts,
VSMCs, and connective tissue cells. PDGF can be present as either homodimer or heterodimer
of A and B chains, which bind to its dimeric receptor composed of all three combinations of 
and  subunits [79]. The cleavage of the PDGF A-chain mRNA by hammerhead ribozyme
attenuated human and rat VSMC growth in vitro [80] [81]. This same strategy, as well as
nanospheres containing antisense ODN against PDGF receptor, inhibited neointimal thickening
and thrombus formation in the rat carotid artery model of balloon angioplasty [82] [83].
Platelet derived endothelial cell growth factor (PD-ECGF)
PD-ECGF (also known as thymidine phosphorylase) stimulates chemotaxis of ECs and
angiogenesis [84, 85]. Gene transfer of PD-ECGF increased the expression of heme oxygenase
and p27kip1 in cultured rat VSMCs, and inhibited neointima formation in balloon-injured rat
carotid arteries [86].
Vascular endothelial growth factor (VEGF)
VEGF is a proangiogenic cytokine that promotes EC growth and survival and also
stimulates monocyte activation and migration [87]. Members of the VEGF family (VEGF-A,
VEGF-B, VEGF-C, VEGF-D, and placenta growth factor) (PlGF) interact with a set of cell
surface receptors (VEGFR-1, VEGFR-2, and VEGFR-3) with varying specificity and function.
Atherosclerosis in several animal models was aggravated by intramuscular injection of
recombinant human VEGF protein [88, 89], and adenovirus-mediated PlGF transfer promoted
macrophage accumulation, endothelial vascular cell adhesion molecule 1 expression and
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neointima formation in collared arteries of hypercholesterolemic rabbits [90]. Consistent with
these findings, blockade of VEGF by soluble VEGF receptor (sFlt-1) gene transfer attenuated
neointima formation after experimental angioplasty, and this correlated with reduced vascular
inflammation and proliferation [91]. It has been suggested that the proatherogenic effect of
VEGF might be due to augmented angiogenesis, since the angiogenesis inhibitors endostatin and
TNP-470 reduced intimal neovascularization and plaque growth in apoE-null mice [92].
In contrast, several animal studies have refuted the notion that VEGF accelerates
atherosclerosis [93, 94], and no evidence of increased atherogenesis in human arteries has been
observed after VEGF-A gene transfer or protein delivery in several clinical trials, including
VEGF in Ischemia for Vascular Angiogenesis Trial (VIVA) [95], Kuopio Angiogenesis Trial
(KAT) [96], and the Euroinject Once Trial [97]. Indeed, a critical review of the literature has led
Khurana et al. to conclude that, although microvessels are present in advanced human atheroma,
angiogenesis and angiogenic factors do not contribute significantly to atherosclerosis and
neointima formation[98].
Finally, it is noteworthy that VEGF has shown beneficial effects against neointimal
thickening induced by mechanical injury. Ectopic VEGF expression using either “naked”
plasmidic DNA or adenoviral vectors accelerates reendothelialization and attenuates neointimal
thickening in animal models of endothelial denudation [99] [100, 101] [102] [103, 104].
Conversely, sequestration of exogenous and/or endogenous VEGF by a VEGF-trap strategy
delayed reendothelialization and significantly increased neointima [103]. Using an external
vascular collar placement model of neointimal thickening in which the endothelium is not
removed, Khurana et al. showed that VEGF164 (VEGF-A) gene transfer inhibited neointimal
formation and macrophage accumulation in the carotid artery of hypercholesterolemic rabbits
[105]. NO production seems to play a key role in the therapeutic effect of VEGF in collar and
angioplasty models, as VEGF induces NO production and treatment with the NO synthase
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inhibitor NG-nitro-L-arginine methyl ester (L-NAME) blocked the reduction in neointimal
thickening elicited by VEGF [106] [104]. As pointed out by Tsutsumi and Losordo, the
controversy over VEGF role in neointimal thikening is certainly not solved and mechanisms to
explain the disparate findings in the literature will continue to be the matter of intense study
[107].
Transfer of VEGF or VEGF-containing fusion genes has been claimed to achieve bone
marrow vascularization, bone growth, and wound healing, and to stimulate angiogenesis as a
treatment for peripheral and myocardial ischemia [311, 312, 313]. Moreover, Isis Pharm Inc has
described the use of antisense ODN targeted to VEGF-1 and VEGFR-2 for the treatment of
hyperproliferative disorders, including cancer, a disease or condition involving angiogenesis, or
rheumatoid arthritis [314, 315].
2.3. Transcription factors involved in cell cycle control
Yin Yang-1 (YY1)
The transcription factor YY1 can repress the expression of many proatherogenic
molecules, including growth factors, hormones, and cytokines. Using an in vitro scraping model
and the in vivo rat balloon angioplasty model of vascular injury, Santiago et al. have shown that
YY1 expression and DNA-binding activity are activated in rat VSMCs after injury [108]. FGF-2
increased YY1 mRNA and protein expression and stimulated YY1 binding and transcriptional
activity, and neutralizing antibodies directed against FGF-2 blocked YY1 induction after injury.
Interestingly, YY1 overexpression inhibits VSMC but not EC proliferation. Unisearch Ltd has
described methods for inhibiting cellular proliferation via YY1 overexpression, as well as
methods of screening for compounds which inhibit cell proliferation and for treating or
preventing cancer, atherosclerosis and restenosis (e. g., by using YY1-coated stents) [318].
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E2F
The transcription factor E2F activates the expression of genes encoding proteins that are
required for nucleotide and DNA biosynthesis (e. g., DNA polymerase , histone H2A, pcna,
thymidine kinase) and of several growth and cell-cycle regulators (e. g., c-myc, pRb, cdc2, cyclin
E, cyclin A) [109]. Neointimal thickening in balloon-injured arteries [110-112], vein grafts [113,
114] [115], and cardiac allografts [116] is reduced by delivering a synthetic ‘decoy’ ODN
containing the optimum E2F DNA target sequence. This strategy leads to the sequestration of
endogenous E2F thus attenuating its interaction with the authentic cis-elements in cellular target
genes. Ahn et al. developed an E2F ‘decoy’ ODN with a circular dumbbell structure (CD-E2F)
and compared its properties with those of conventional phosphorothioated E2F ‘decoy’ ODN
(PS-E2F) [117]. CD-E2F displayed more stability and stronger antiproliferative activity than PSE2F in cultured VSMCs, and was more effective at inhibiting neointimal thickening in vivo. It
has been recently shown that the combination of hammerhead ribozymes targeted against cyclin
E and E2F1 downregulates coronary VSMC proliferation more efficiently than either gene
transfer alone [118].
c-myc and c-myb
Several “immediate-early” genes (e. g., c-fos, c-jun, c-myc, c-myb, egr-1) are induced in
serum-stimulated VSMCs, and their overexpression can promote VSMC proliferation. Cultures
of VSMCs obtained from atheromatous plaques exhibit higher levels of c-myc mRNA than
VSMCs from normal arteries [119], and arterial injury induced the expression of several
“immediate-early” genes [120]. Antisense ODNs against c-myc and c-myb reportedly inhibited in
a sequence-specific manner VSMC proliferation in vitro [121-129] and neointima formation
16
after balloon angioplasty [121, 125, 129-132] [133] and vein grafting[134]. However, it has been
claimed that these inhibitory effects may be mediated by a nonspecific mechanism [135-139].
NFB
The transcription factor NFB activates the expression of cytokines and adhesion
molecules involved in several diseases. Transfection of NFB ‘decoy’ ODN reduced
angioplasty-induced neointimal thickening in rat carotid [140] and porcine coronary [141]
arteries, but not in New Zealand rabbit iliac arteries [142]. Adenoviral gene transfer of an IB
mutant that inhibits NFB upregulated the growth suppressors p21Cip1 and p27Kip1 and attenuated
intimal lesion formation after balloon injury in rat carotid arteries [143].
Activator protein-1 (AP-1)
Both the expression and DNA-binding activity of transcription factors of the AP-1 family
are induced in the rat carotid artery model of angioplasty coinciding with high proliferative
activity [120]. Transfection of AP-1 ‘decoy’ ODN into cultured human VSMCs significantly
reduced PDGF-dependent increase in cell number and TGF-1 production [144], and attenuated
neointimal thickening when applied at the site of balloon angioplasty in rabbit carotid [144] and
minipig coronary [145] arteries. Circular dumbbell AP-1 ‘decoy’ ODN was more effective than
conventional phosphorothioated AP-1 ‘decoy’ ODN at inhibiting the proliferation of cultured
VSMCs and neointimal hyperplasia after balloon angioplasty to the rat carotid artery [146].
GAX
Expression of the homeobox gene Gax is rapidly downregulated in vitro upon growth
factor stimulation of serum-starved VSMCs and after balloon angioplasty to the rat carotid artery
[147, 148]. Moreover, Gax overexpression inhibited VSMC proliferation in vitro and attenuated
neointimal thickening in balloon-injured rat carotid arteries in a p21Cip1-dependent manner [149,
17
150]. Percutaneous delivery [151] and adenovirus-mediated [152] transfer of the Gax gene also
inhibited vessel stenosis in a rabbit model of balloon angioplasty.
FOXO proteins
FOXO proteins are members of the forkhead box transcription factor family [153]. These
proteins are inactivated by growth factor-induced phosphorylation, which leads to their nuclear
exclusion and downregulation of target genes required for growth arrest (e. g. p27Kip1).
Overexpression of constitutively active, nonphosphorylatable FOXO mutant TM-FMHRL1
significantly increased p27Kip1 expression and apoptosis, and attenuated proliferative index and
neointimal thickening in balloon-injured rat carotid arteries [154] [155].
2.4. Miscellaneous
Prostacyclin
Prostacyclin is an endothelium-derived factor with vasoactive and antiproliferative
properties. The gene transfer of cyclooxygenase-1 [156] and prostacyclin synthase [157], both of
which are implicated in the synthesis of prostacyclin, can attenuate neointimal hyperplasia after
angioplasty. Medicinal compositions containing prostacyclin synthase gene have been claimed
for gene therapy of cancer and intimal proliferation [319].
12-Lipoxygenase
12-Lipoxygenase products of arachidonate metabolism promote growth and chemotaxis
on VSMCs, and ribozyme against this enzyme prevents intimal hyperplasia in balloon-injured rat
carotid arteries [158].
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Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA)
SERCA gene transfer reduces VSMC proliferation and neointima formation in balloon
injured rat carotid artery [159].
Calcitonin gene-related peptide (CGRP)
The intramuscular transfection of CGRP reduces neointima formation after balloon injury
in rat aorta [160]. This effect is associated with an increase in iNOS and the growth suppressor
p53 proteins, and a reduction in PCNA and Bcl-2 proteins.
3. Antiproliferative gene therapy clinical trials for human occlusive vascular
disease
Restenosis after successful PTA and graft failure after by-pass surgery are complications
that typically develop over a short period of time after intervention (e. g., 2-12 months) and thus
remain the major limitation of these revascularization strategies. In this section, we discuss the
results of gene therapy clinical trials targeting E2F, c-myc and VEGF (Table).
3.1. E2F decoy
Surgical bypass of occluded arteries with autologous vein grafts has been widely used to
revascularize ischemic territories. However, graft failure as a consequence of neointimal
hyperplasia may run as high as 50% within 5 years. Based on the encouraging results of the E2F
‘decoy’ strategy in animal models of balloon angioplasty and graft atherosclerosis (see above),
Mann and coworkers initiated the first Project of Ex-vivo Vein graft Engineering via
19
Transfection (PREVENT I) trial, a single-centre, randomized, controlled gene therapy study that
included 41 patients undergoing bypass surgery for the treatment of peripheral arterial occlusions
[161]. Patients were randomly assigned untreated (n=16), E2F-‘decoy’-ODN-treated (n=17), or
scrambled-ODN-treated (n=8) human infrainguinal vein grafts. Ex vivo delivery of ODNs was
achieved intraoperatively via pressure-mediated transfection, which resulted in a 70-74%
decrease in the level of PCNA and c-myc mRNA expressed by the VSMCs in the grafted vein,
and a statistically significant reduction in primary graft failure compared to control groups.
Following to this pilot trial, a randomized, double-blinded, placebo controlled Phase IIb trial
(PREVENT II) was carried out in patients undergoing coronary artery bypass graft (CABG)
surgery. The results of quantitative coronary angiography and intravascular ultrasound (IVUS)
showed larger patency and inhibition of neointimal thickening in treated patients at 12 months
after intervention [4].
PREVENT III, a randomized, double-blind, multicenter clinical trial, has been designed
to evaluate the safety and efficacy of E2F ‘decoy’ (edifoligide) in a population of approximately
1,400 patients with critical limb ischemia undergoing infrainguinal bypass for peripheral arterial
disease [162]. The primary outcome measure will be the time to occurrence of non-technical
graft failure resulting in either graft revision or major amputation at 12 months after enrollment.
PREVENT IV is a phase-III, multicenter, randomized double-blind placebo-controlled
trial that has been designed to establish whether ex vivo treatment of autologous vein grafts with
edifoligide in patients undergoing initial CABG surgery can improve graft patency and reduce
adverse cardiac events, morbidity and mortality [163]. A total of 1,920 patients (80% of enrolled
patients) either died (n=91) or underwent follow-up angiography (n=1,829). It was found that
edifoligide had no effect on the primary end-point of per patient vein graft failure, or on the
incidence of major adverse cardiac events at 1 year. The authors concluded that "Longer-term
follow-up and additional research are needed to determine whether edifoligide has delayed
20
beneficial effects, to understand the mechanisms and clinical consequences of vein graft failure,
and to improve the durability of CABG surgery."
3.2. c-myc
Roque et al. assessed the pharmacokinetics and clinical safety of ascending doses of cmyc antisense ODN (LR-3280) administered after PTCA [164]. Seventy eight patients were
randomized to receive standard treatment without (n = 26) or with escalating doses (1 to 24 mg)
of LR-3280 (n = 52), administered into target vessel through a guiding catheter. The peak plasma
concentrations of LR-3280 occurred at 1 minute and decreased rapidly after approximately 1
hour, with little LR-3280 detected in the urine between 0-6 hours and 12-24 hours. The
intracoronary administration of LR-3280 was well tolerated at doses up to 24 mg and produced
no adverse effects in dilated coronary arteries, thus providing the basis for the evaluation of local
delivery of c-myc antisense ODN for the prevention of human vasculoproliferative disease.
Kutryk et al. reported the results of the Investigation by the Thoraxcenter of Antisense
DNA using Local delivery and IVUS after Coronary Stenting (ITALICS) trial [165]. This
randomized, placebo controlled study was designed to determine the efficacy of c-myc antisense
ODN at inhibiting in-stent restenosis. Eighty-five patients were randomly assigned to receive
either c-myc antisense ODN or saline vehicle by intracoronary local delivery after coronary stent
implantation. Follow-up included the percent neointimal volume obstruction measured by IVUS,
clinical outcome and quantitative coronary angiography. There was no reduction in either the
neointimal volume obstruction or the angiographic restenosis rate after treatment with 10 mg of
phosphorothioate-modified ODN directed against c-myc as demonstrated by the analysis of 77
patients. In contrast, the randomized AVAIL trial demonstrated both safety and long-term
efficacy at reducing neointima formation in a small cohort of coronary artery disease patients
receiving locally c-myc antisense ODN at the time of angioplasty [166].
21
3.3. VEGF
Inhibition of restenosis after angioplasty by hydrogel catheter delivery of naked VEGF165
has been observed in a pilot human trial [167]. Therapeutic angiogenesis is another potential
application of VEGF gene therapy. This strategy, which consists of promoting collateral vessel
formation in ischemic tissues (e. g. to treat myocardial infarction, peripheral artery disease,
critical limb ischemia), has been discussed elsewhere [168]. In general, the therapeutic
angiogenesis clinical trials VIVA, KAT and Euroinject Once have shown that VEGF gene
transfer or VEGF protein delivery can be performed safely and does not increase neither
atherogenesis nor restenosis after angioplasty [95] [96] [97] [96, 168-170].
4. Expert opinion
Excessive cellular proliferation contributes to neointimal thickening in the setting of
atherosclerosis, in-stent restenosis, and vessel graft failure. The analysis of several animal models
suggests that neointimal hyperplasia prevails at the onset of atherogenesis. Because patients
frequently exhibit advanced atherosclerotic plaques when first diagnosed and cell proliferation is
also likely to peak at the early stages of human atheroma development, the potential benefit of
antiproliferative strategies for the treatment of human atherosclerosis is doubtful. Indeed, the
cytostatic approaches used so far in the setting of vasculoproliferative disease have focused on
restenosis and late vessel graft failure after bypass surgery, during which neointimal hyperplasia
is spatially localized and develops over a short period of time (typically 2-12 months).
Cytostatic gene therapy approaches have consisted of gain- and loss-of-function of negative
and positive cell cycle regulatory genes, respectively. These include CDKs, cyclins, growth
22
factors and their receptors, transcription factors, and growth suppressors. Generally,
antiproliferative gene therapy strategies have shown efficacy at limiting neointimal thickening in
animal models of angioplasty and vessel graft failure. While recent clinical trials based on the use
of E2F ‘decoy’ and c-myc antisense ODN gene therapy for the treatment of these disorders have
demonstrated safety, whether they exhibit long-term efficacy remains unknown. Thus, cytostatic
gene therapy for occlusive vascular disease, like in other areas of clinical research, has provided
very little in terms of positive results. Regardless of the final outcome of ongoing clinical trials,
further studies are required to override the current practical barriers and limitations placed on
most studies before gene therapy strategies exhibit wide application in clinic. These should
include improvement of transgene expression, the clarification of safety issues (e. g., achieving
tissue-specific activity and minimising vector-related immunology), and development of better
gene delivery vectors. Moreover, additional research is essential to continue to unravel the
molecular mechanisms governing pathological proliferation of neointimal cells to identify novel
therapeutic targets.
ACKNOWLEDGEMENTS
We apologize to many colleagues whose primary work has not been directly cited due to
space constraints. We thank María J. Andrés-Manzano for the preparation of figures. Work in the
laboratory of V. A. is supported by grants from Instituto de Salud Carlos III (Red de Centros
RECAVA, C03/01), from the Spanish Ministry of Education and Science and the European
Regional Development Fund (SAF2004-03057), and from Laboratorios INDAS, S. A:
23
Table: Gene therapy clinical trials for vascular proliferative disease based on cytostatic
strategies
Trial
PREVENT
I
Design
Randomized,
double-blinded,
single center (41
patients)
Strategy
E2F decoy
ODN
ex vivo
transfection
of vein graft
Disease
Autologous
vein graft
failure after
peripheral
artery bypass
PREVENT
II
Randomized
multicenter,
double-blinded,
placebocontrolled
E2F decoy
ODN
ex vivo
transfection
of vein graft
PREVENT
III
Phase 3
Randomized
multicenter
double-blinded,
multicenter
clinical trial
(approx. 1400
patients)
Phase 3
Randomized
multicenter
double-blinded,
multicenter
clinical trial
(approx 1900
patients)
Randomized,
placebocontrolled
(85 patients)
E2F decoy
ODN
ex vivo
transfection
of vein graft
Autologous
vein
graft failure
after
coronary artery
bypass
Autologous
vein
graft failure
after
peripheral
artery bypass
PREVENT
IV
ITALICS
AVAIL
VIVA
Randomized
randomized,
double-blinded,
placebocontrolled study
(15 patients)
double-blind,
placebocontrolled trial
Outcome
Refs.
70-74% decreases in [161]
the level of positive
cell cycle regulators
expressed by VSMCs
in the vein, and
reduction in primary
graft failure
Larger patency and
[4]
inhibition neointimal
thickening
[162]
E2F decoy
ODN
ex vivo
transfection
of vein graft
Autologous
vein
graft failure
after
coronary artery
bypass
No beneficial effect
[163]
on vein graft failure,
or on the incidence of
major adverse cardiac
events at 1 year.
c-myc
antisense
ODN delivery
after stent
implantation
c-myc
antisense
ODN
In-stent
coronary
restenosis
No reduction in
angiographic
restenosis rate
[165]
In-stent
coronary
restenosis
Reduction of
neointima formation
[166]
VEGF
plasmid/liposo
me gene
transfer after
PTCA
intracoronary
and
intravenous
Myocardial
ischemia
VEGF gene transfer
performed after
PTCA is safe and
well torelated
[169]
Myocardial
ischemia
No evindence of
increased
atherogenesis
[95]
24
(178 patients)
KAT
Euroinject
Once
randomized,
placebocontrolled,
double-blind
phase II study
(103 patients)
phase II
randomized
double-blind trial
(80 patients)
infusions of
rhVEGF gene
transfer
CatheterMyocardial
based
ischemia
intracoronary
VEGF gene
transfer
percutaneous Myocardial
intramyocardi ischemia
al plasmid
VEGF165 gene
transfer
No evindence of
increased
atherogenesis
[96]
No evindence of
increased
atherogenesis
[97]
PREVENT: Project of ex-vivo vein graft engineering via transfection
ITALICS:
Investigation by the thoraxcenter of antisense DNA using local delivery and IVUS
after coronary stenting
VIVA: Vascular endothelial growth factor in Ischemia for Vascular Angiogenesis.
KAT: Kuopio Angiogenesis Trial
25
FIGURE LEGENDS
Figure 1. A. Cell cycle progression is controlled by several CDKs that associate with
regulatory subunits called cyclins. Mitogenic stimuli activate CDK/cyclin holoenzymes,
thus causing hyperphosphorylation of the retinoblastoma protein (pRb) and related
pocket proteins from mid G1 to mitosis, and inducing the transactivation of genes with
functional E2F-binding sites (e. g., growth and cell-cycle regulators and genes encoding
proteins that are required for nucleotide and DNA biosynthesis). B. Proteins of the CKI
family cause growth suppression by interacting with and inhibiting CDK/cyclin activity.
CKIs of the Cip/Kip family (p21Cip1, p27Kip1 and p57Kip2) bind to and inhibit a wide
spectrum of CDK/cyclin holoenzymes, while members of the Ink4 family (p15Ink4b,
p16Ink4a, p18Ink4c, p19Ink4d) are specific for cyclin D-associated CDKs.
Figure 2. p53 regulates the expression of genes that control cell cycle progression,
apoptosis, DNA repair and other functions required for an appropriate cellular response
to different forms of stress.
26
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