Gene Therapy Antiproliferative Strategies Against Cardiovascular Disease
Marisol Gascón-Irún, Silvia M. Sanz-González and Vicente Andrés
Laboratory of Vascular Biology, Department of Molecular and Cellular Pathology and Therapy,
Instituto de Biomedicina de Valencia, Spanish Council for Scientific Research (CSIC), Valencia,
Spain.
Corresponding author:
Vicente Andrés
Tel: +34-96-3391752
FAX: +34-96-3690800
E-mail: vandres@ibv.csic.es
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ABSTRACT
Excessive cellular proliferation is thought to contribute to the pathogenesis of several
forms of cardiovascular disease (e. g., atherosclerosis, restenosis after angioplasty, and vessel
bypass graft failure). Therefore, candidate targets for the treatment of these disorders 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.
Importantly, animal models of atherosclerosis have demonstrated an inverse correlation between
neointimal cell proliferation and atheroma size, suggesting that excessive cell growth prevails at
the onset of atherogenesis. Cell growth may also predominate at the onset of human
atherosclerosis. Thus, given that affected humans often exhibit advanced atherosclerotic plaques
when first diagnosed, the potential benefit of antiproliferative strategies for the treatment of
atherosclerosis in clinic is doubtful.
The antiproliferative approaches used so far in the setting of vascular obstructive disease
have focused on restenosis and graft atherosclerosis, during which neointimal hyperplasia is
spatially localized and develops over a short period of time (typically 2-12 months). Vascular
interventions, both endovascular and open surgical, allow minimally invasive, easily monitored
gene delivery. Thus, gene therapy strategy are emerging as an attractive approach for the
treatment of vascular proliferative disease. In this review, we will discuss the use of gene therapy
strategies
against cellular proliferation in animal models and clinical trials of cardiovascular
disease.
KEY WORDS: atherosclerosis, restenosis, bypass graft failure, cell cycle, gene therapy
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LIST OF ABBREVIATIONS: apoE, apolipoprotein E; AP-1, activator protein-1; BrdU, 5bromodeoxyuridine; CDK, cyclin-dependent kinase; CKI, CDK inhibitory
protein; EC, endothelial cell; ERK, extracellular signal-regulated kinase;
IVUS, intravascular ultrasound; JNK, c-jun NH2-terminal protein kinase;
MAPK, mitogen-activated protein kinase; ODN, oligodeoxynucleotide;
PCNA, proliferating cell nuclear antigen; PDGF, platelet-derived growth
factor; pRb, retinoblastoma protein; PTCA, percutaneous transluminal
angioplasty; SAPK, stress-activated protein kinase; TGF-transforming
growth factor-; VSMC, vascular smooth muscle cell.
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I. Introduction
II. Preclinical studies
IIA. Antisense approach
IIA1. CDKs and cyclins
IIA2. Mitogen-responsive nuclear factors that promote cell growth
IIB. Ribozymes
IIC. Transcription factor ‘decoy’ strategies
IIC1. E2F
IIC2. AP-1
IID. Overexpression of growth suppressors
IID1. CKIs
IID2. p53
IID3. pRb
IID4. GATA-6
IID5. GAX
IIE. Overexpression of transdominant negative mutants of positive cell cycle
regulators.
IIE1. Ras
IIE2. MAPKs
III. Clinical studies
IIIA. E2F ‘decoy’
IIIB. c-myc antisense ODN
IV. Conclusions
Acknowledegments
References
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I. Introduction
Large-scale clinical trials conducted over the last decades have allowed the identification
of independent risk factors that increase the prevalence and severity of atherosclerosis (e. g.,
hypercholesterolemia, hypertension, smoking). Cardiovascular risk factors initiate and perpetuate
an inflammatory response within the injured arterial wall that promotes the development of
atherosclerotic plaques (Dzau et al., 2002; Lusis, 2000; Ross, 1999; Steinberg, 2002) (Fig. 1).
Chemokines and cytokines secreted by leukocytes that accumulate within the injured arterial
wall promote their own proliferation, as well as the growth and migration of the underlying
vascular smooth muscle cells (VSMCs) (Fig. 2). This inflammatory response also plays a critical
role during restenosis after angioplasty and graft atherosclerosis. Thus, understanding the
molecular mechanisms that control hyperplastic growth of vascular cells should help develop
novel therapeutic strategies for the treatment of vascular obstructive disease.
Although arterial cell proliferation occurs in animal models during all phases of
atherogenesis (Cortés et al., 2002; Díez-Juan and Andrés, 2001; Ross, 1999), studies with
hyperlipidemic rabbits have shown an inverse correlation between atheroma size and cellular
proliferation within the atheromatous plaque (McMillan and Stary, 1968; Rosenfeld and Ross,
1990; Spraragen et al., 1962). Experimental angioplasty is also characterized by abundant
proliferation of VSMCs, followed by the reestablishment of the quiescent phenotype, typically
within 2-4 weeks (Andrés, 1998; Bauters and Isner, 1997; Libby and Tanaka, 1997). These
animal studies suggest that vascular cell proliferation prevails at the onset of atherogenesis and
restenosis.
Expression of proliferation markers in human primary atheromatous plaques and restenotic
lesions has been well documented (Burrig, 1991; Essed et al., 1983; Gordon et al., 1990; Katsuda
et al., 1993; Kearney et al., 1997; Nobuyoshi et al., 1991; O'Brien et al., 1993; O'Brien et al.,
2000; Orekhov et al., 1998; Rekhter and Gordon, 1995; Tanner et al., 1998; Veinot et al., 1998;
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Wei et al., 1997). However, controversy exists regarding the magnitude of the proliferative
response, ranging from a very low index of cell proliferation (Gordon et al., 1990; Katsuda et al.,
1993; O'Brien et al., 1993; O'Brien et al., 2000; Rekhter and Gordon, 1995; Veinot et al., 1998)
to abundance of dividing cells (Essed et al., 1983; Kearney et al., 1997; Nobuyoshi et al., 1991;
Pickering et al., 1993). Aside from methodological issues (e. g., differences in the fixatives used
for tissue preservation, antigen accessibility, diversity of proliferation markers analyzed in these
studies), some of the reported variance with regard to the issue of cell proliferation might relate
to differences in the arteries being analyzed (i. e., peripheral, coronary and carotid arteries) and
variance in the stage of atherogenesis at the time of tissue harvesting (Isner, 1994).
The cell types that undergo cell proliferation within human atherosclerotic tissue include
VSMCs, leukocytes and endothelial cells (ECs) (Burrig, 1991; Gordon et al., 1990; Katsuda et
al., 1993; O'Brien et al., 1993; Orekhov et al., 1998; Rekhter and Gordon, 1995; Veinot et al.,
1998). Histological examination in 20 patients undergoing antemorten coronary angioplasty
revealed that the extent of intimal proliferation was significantly greater in lesions with evidence
of medial or adventitial tears than in lesions with no or only intimal tears (Nobuyoshi et al.,
1991). Human carotid artery primary atherosclerotic tissue retrieved by endarterectomy surgery
displayed greater proliferative activity in the intimal lesion versus the underlying media (Rekhter
and Gordon, 1995). Moreover, monocyte/macrophage proliferation predominated in the intima
(46% versus 9.7% -actin immunoreactive VSMCs, 14.3% ECs, 13.1% T lymphocytes),
whereas VSMC proliferation prevailed in the media (44.4% versus 20% ECs, 13.0%
monocyte/macrophages, and 14.3% T lymphocytes). It is also noteworthy that cell proliferation
in human peripheral and coronary ateries is greater in restenotic versus primary lesions (O'Brien
et al., 1993; O'Brien et al., 2000; Pickering et al., 1993). Furthermore, cultured VSMCs from
human advanced primary stenosing disclosed lower proliferative capacity than cells from fresh
restenosing lesions (Dartsch et al., 1990). Thus, similar to the situation in animal models,
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proliferation during human atherosclerosis and restenosis might peak at the onset of these
pathologies and then progressively decline.
Cell cycle progression is controlled by several cyclin-dependent kinases (CDKs) that
associate with regulatory cyclins (Morgan, 1995) (Fig. 3). Active CDK/cyclin holoenzymes
hyperphosphorylate the retinoblastoma protein (pRb) and the related pocket proteins p107 and
p130 from mid G1 to mitosis. Phosphorylation of pRb and related pocket proteins contributes to
the transactivation of genes with functional E2F-binding sites, including several growth and cellcycle regulators (i.e., c-myc, pRb, cdc2, cyclin E, cyclin A), and genes encoding proteins that are
required for nucleotide and DNA biosynthesis (i. e., DNA polymerase , histone H2A,
proliferating cell nuclear antigen, thymidine kinase) (Dyson, 1998; Lavia and Jansen-Durr, 1999;
Stevaux and Dyson, 2002). Interaction of CDK/cyclins with CDK inhibitory proteins (CKIs)
attenuates CDK activity and promotes growth arrest (Philipp-Staheli et al., 2001). 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 (p16Ink4a, p15Ink4b, p18Ink4c, p19Ink4d) are specific
for cyclin D-associated CDKs. Mitogenic and antimitogenic stimuli affect the rates of synthesis
and degradation of CKIs, as well as their redistribution among different CDK/cyclin pairs
(Philipp-Staheli et al., 2001). For example, p27Kip1 promotes the assembly of CDK4/cyclin D
complexes by binding to them, thus facilitating CDK2/cyclin E activation through G1/S phase.
VSMC proliferation in the balloon-injured rat carotid artery is associated with a temporally
and spatially coordinated expression of CDKs and cyclins (Braun-Dullaeus et al., 2001; Wei et
al., 1997). Importantly, augmented expression of these factors is associated with an increase in
their kinase activity (Abe et al., 1994; Wei et al., 1997), demonstrating the assembly of
functional CDK/cyclin holoenzymes in the injured arterial wall. Expression of CDK2 and cyclin
E was also detected in human VSMCs within atherosclerotic and restenotic tissue (Ihling et al.,
1999; Kearney et al., 1997; Wei et al., 1997), suggesting that induction of positive cell-cycle
7
control genes is a hallmark of vascular proliferative disease in human patients.
In the following sections, we will discuss the use of gene therapy strategies targeting
cellular proliferation in preclinical (Table 1) and clinical studies (Table 2) related to
cardiovascular disease.
II. Preclinical studies
Antiproliferative gene therapy strategies designed for the treatment of experimental
cardiovascular disease include the following: 1) inactivation of positive cell cycle regulators (e.
g., CDK/cyclins, protooncogenes, E2F, growth factors) by antisense approaches, ribozymes, and
transcription factor ‘decoy’ strategies (Fig. 4), 2) overexpression of negative regulators of cell
growth (e. g., CKIs, p53, pRb, GAX, and GATA-6), and 3) overexpression of transdominant
negative mutants of positive cell cycle regulators (e. g., Ras, mitogen-activated protein kinases).
IIA. Antisense approach
The gene of interest is inactivated by using a synthetic antisense oligodeoxynucleotide
(ODN) that hybridizes in a complementary fashion and stoicheometrically with the target
mRNA.
IIA1. CDKs and cyclins
The efficacy of antisense ODN strategies targeting CDKs and cyclins to reduce neointimal
lesion formation has been demonstrated in several animal models of balloon angioplasty. These
studies include antisense oligodeoxynucleotides against CDK2 (Abe et al., 1994; Morishita et al.,
1994a), CDC2 (Abe et al., 1994; Morishita et al., 1993; Morishita et al., 1994b) and cyclin B1
(Morishita et al., 1994b). Interestingly, cotransfection of antisense ODN against CDC2 kinase
and cyclin B1 resulted in further inhibition of neointima formation, as compared to blockade of
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either gene target alone (Morishita et al., 1994b). Of note, Morishita et al. reported sustained
inhibition of neointima formation in the rat carotid balloon-injury model after a single
intraluminal molecular delivery of combined CDC2 and proliferating cell nuclear antigen
(PCNA) antisense ODNs (Morishita et al., 1993), whereas this approach had no effect in the
coronary arteries of pigs after balloon angioplasty (Robinson et al., 1997). Downregulation of
cyclin G1 expression by retrovirus-mediated antisense gene transfer inhibited VSMC
proliferation and neointima formation after balloon angioplasty (Zhu et al., 1997). Attenuated
graft atherosclerosis has been also observed upon inactivation of CDC2/PCNA (Mann et al.,
1995; Miniati et al., 2000 2000) and CDK2 (Suzuki et al., 1997) with antisense ODN.
IIA2. Mitogen-responsive nuclear factors that promote cell growth
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 in vitro
(Bennett et al., 1994b; Brown et al., 1992; Campan et al., 1992; Castellot et al., 1985; Gorski and
Walsh, 1995; Kindy and Sonenshein, 1986; Reilly et al., 1989; Rothman et al., 1994). VSMCs
cultured from atheromatous plaques present higher levels of c-myc mRNA than VSMCs from
normal arteries (Parkes et al., 1991), and arterial injury induced the expression of several
“immediate-early” genes (Lambert et al., 2001; Miano et al., 1990; Miano et al., 1993; Sylvester
et al., 1998). Antisense ODNs against c-myc and c-myb reportedly inhibited in a sequencespecific manner both VSMC proliferation in vitro (Bennett et al., 1994a; Biro et al., 1993; Brown
et al., 1992; Ebbecke et al., 1992; Gunn et al., 1997; Pukac et al., 1990; Shi et al., 1994a; Shi et
al., 1993; Simons and Rosenberg, 1992), and neointima formation after angioplasty (Bennett et
al., 1994a; Gunn et al., 1997; Kipshidze et al., 2001; Kipshidze et al., 2002; Shi et al., 1994b;
Simons et al., 1992) and vein grafting (Mannion et al., 1998) in vivo. However, these inhibitory
effects may be mediated by a nonantisense mechanism (Burgess et al., 1995; Chavany et al.,
1995; Guvakova et al., 1995; Villa et al., 1995; Wang et al., 1996).
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It has been recently shown that nanospheres containing antisense ODN against PDGF
receptor inhibit neointimal thickening in the rat carotid model of balloon angioplasty (CohenSacks et al., 2002).
IIB. Ribozymes
Ribozymes represent a unique class of RNA molecules that catalytically cleave the specific
target RNA, thus resulting in targeted gene inactivation. Su et al. designed a DNA-RNA
chimeric hammerhead ribozyme targeted to human transforming growth factor-1 (TGF-1) that
significantly inhibited angiotensin II-stimulated TGF-1 mRNA and protein expression in
human VSMCs, and efficiently inhibited the growth of these cells (Su et al., 2000). Likewise,
cleavage of the platelet-derived growth factor (PDGF) A-chain mRNA by hammerhead
ribozyme attenuated human and rat VSMC growth in vitro (Hu et al., 2001a; Hu et al., 2001b)
and inhibited neointima formation in the rat carotid artery model of balloon injury (Kotani et al.,
2003).
Studies using experimental models of angioplasty provided the first evidence that
ribozymes might represent useful tools in cardiovascular therapy. Frimerman et al. reported the
efficacy of chimeric hammerhead ribozyme to PCNA in reducing stent-induced stenosis in a
porcine coronary model (Frimerman et al., 1999), and ribozyme strategy against TGF-1
inhibited neointimal formation after balloon injury in the rat carotid artery model (Yamamoto et
al., 2000). 12-Lipoxygenase products of arachidonate metabolism have growth and chemotactic
effects in vascular smooth muscle cells, and ribozyme against this enzyme prevents intimal
hyperplasia in balloon-injured rat carotid arteries (Gu et al., 2001).
IIC. Transcription factor ‘decoy’ strategies
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This approach consists of delivering a double-stranded ODN corresponding to the
optimum DNA target sequence of the transcription factor of interest, thus leading to the
sequestration of the specific trans-acting factor and attenuation of its interaction with the
authentic cis-elements in cellular target genes.
IIC1. E2F
E2F participates in the transcriptional activation of genes encoding proteins that are
required for nucleotide and DNA biosynthesis (e. g., DNA polymerase , histone H2A, pcna,
thymidine kinase) (Dyson, 1998; Lavia and Jansen-Durr, 1999) and in several growth and cellcycle regulators (e. g., c-myc, pRb, cdc2, cyclin E, cyclin A).
Experimental neointimal thickening in balloon-injured arteries (Morishita et al., 1995;
Nakamura et al., 2002), vein grafts (Ehsan et al., 2001; Mann et al., 1997), and cardiac allografts
(Kawauchi et al., 2000) is prevented by the use of a synthetic ‘decoy’ ODN containing an E2F
consensus binding site that inactivates the transcription factor E2F. Ahn et al. developed a novel
E2F ‘decoy’ ODN with a circular dumbbell structure (CD-E2F) and compared its properties with
those of conventional phosphorothioated E2F ‘decoy’ ODN (PS-E2F) (Ahn et al., 2002a). CDE2F displayed more stability and stronger antiproliferative activity than PS-E2F when assayed in
cultured VSMCs, and was more effective in inhibiting neointimal formation in vivo.
IIC2. Activator protein-1 (AP-1)
Cell proliferation in the rat carotid artery model of angioplasty correlated with elevated
expression and high DNA-binding activity of transcription factors of the AP-1 family (Andrés et
al., 2001; Hu et al., 1997; Miano et al., 1990; Miano et al., 1993; Sylvester et al., 1998). Under
conditions of PDGF stimulation, AP-1 ‘decoy’ ODN delivery into cultured human VSMCs
significantly reduced cell number and TGF-1 production (Kume et al., 2002), and attenuated
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neointimal thickening when applied at the site of balloon angioplasty in rabbit carotid artery
(Kume et al., 2002) and minipig coronary arteries (Buchwald et al., 2002). Circular dumbbell
AP-1 ‘decoy’ ODN was more effective in inhibiting the proliferation of VSMCs in vitro and
neointimal hyperplasia in vivo compared to conventional phosphorothioated AP-1 decoy ODN,
(Ahn et al., 2002b).
IID. Overexpression of growth suppressors
IID1. CKIs
The efficacy of CKIs in inhibiting CDK activity and cell cycle progression has been widely
documented in a variety of normal and tumour cells in vitro. The first evidence that p21Cip1 and
p27Kip1 may function as negative regulators of neointimal hyperplasia was suggested in animal
studies showing the upregulation of these CKIs at late time points following balloon angioplasty,
coinciding with the restoration of the quiescent phenotype after the initial proliferative wave
(Chen et al., 1997; Tanner et al., 1998). The protective role of p27Kip1 against neointimal
thickening has been demonstrated in hypercholesterolemic apolipoprotein E (apoE)-deficient
mice, in which genetic inactivation of p27Kip1 accelerated atherogenesis in a dose-dependent
manner (Díez-Juan and Andrés, 2001). However, neointimal hyperplasia after mechanical
damage of the arterial wall was similar in wild-type and p27Kip1-null mice (Roque et al., 2001b).
Redundant roles between p21Cip1 and p27Kip1, or compensatory increase in p21Cip1 expression (or
other CKIs) might account for the lack of phenotype of p27 Kip1-null mice in the setting of
mechanical arterial injury.
Several studies have suggested a role of CKIs in establishing regional phenotypic variance
in VSMCs from different vascular beds. Using human VSMCs isolated from internal mammary
artery and saphenous vein, Yang et al. suggested that sustained p27 Kip1 expression in spite of
12
growth stimuli may contribute to the resistance to growth of VSMCs from internal mammary
artery and to the longer patency of arterial versus venous grafts (Yang et al., 1998). Likewise,
different expression of p15Ink4b and p27Kip1 has been correlated with distinct proliferative
response of intimal and medial VSMCs towards basic fibroblast growth factor (bFGF or FGF2)
(Olson et al., 2000). Intrinsic differences in the regulation of p27Kip1 might also play an important
role in creating variance in the proliferative and migratory capacity of VSMCs isolated from
different vascular beds, which might in turn contribute to establishing regional variability in
atherogenicity (Castro et al., 2003).
Tanner et al have reported more frequent expression of p27Kip1 and p21Cip1 within regions of
human coronary atheromas not undergoing proliferation (Tanner et al., 1998). Concordant
expression of TGF- receptors I and II in virtually all cells positive for p27 Kip1 within human
atherosclerotic plaques indicates that TGF-1 present in these lesions may contribute to p27Kip1
upregulation (Ihling et al., 1999). Moreover, coexpression of p53 and p21Cip1 in human carotid
atheromatous plaque cells that revealed lack of proliferation markers suggests that induction of
p21Cip1 may occur via transcriptional activation by p53 (Ihling et al., 1997).
Ectopic expression of p21Cip1 and p27Kip1, but not p16Ink4a, significantly reduced neointimal
thickening in several animal models of angioplasty (Chang et al., 1995a; Chen et al., 1997;
Condorelli et al., 2001; Tanner et al., 2000; Ueno et al., 1997a; Yang et al., 1996).
Overexpression of p21Cip1 also attenuated neointimal lesion formation in a rabbit model of vein
grafting (Bai et al., 1998).
IID2. p53
p53 is a transcription factor that functions as a tumor suppressor displaying both
antiproliferative and proapoptotic actions. These effects result from complex regulatory
networks, including transcriptional activation of antiproliferative and proapoptotic genes (e. g.,
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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). Increased VSMC proliferation has been shown as a result of antisense
p53 ODN transfection (Aoki et al., 1999; Matsushita et al., 2000), and p53 gene transfer has the
opposite effect (Yonemitsu et al., 1998). Mayr et al showed a higher rate of proliferation and
migration of VSMCs isolated from p53-deficient mice than its wild-type counterparts (Mayr et
al., 2002). Consistent with these findings, early migration and proliferation of VSMCs happened
in explanted porcine tunica media tissue after mitogen-induced downregulation of p53
(Rodriguez-Campos et al., 2001).
p53 deficiency has been demonstrated to have a proatherogenic effect in studies of genetic
inactivation in hypercholesterolemic apoE and apoE*3-Leiden mice, although the relative
contribution of increased cellular proliferation and decreased apoptosis in these animal models
remains obscure (Guevara et al., 1999; van Vlijmen et al., 2001). Mice deficient for p53 also
disclosed accelerated vein graft atherosclerosis (Mayr et al., 2002). Regarding human
atherosclerosis, p53 is overexpressed but not mutated in human atherosclerotic tissue (Iacopetta
et al., 1995), and lack of proliferation markers in vascular cells coexpressing p53 and p21Cip1
within advanced human atherosclerotic lesions suggests that transcriptional activation of the
p21Cip1 gene by p53 may be a protective mechanism against excessive vascular cell growth
(Ihling et al., 1997).
p53 appears to play an important role in the pathogenesis of restenosis, as suggested by
both animal and human studies. Transfection of antisense p53 ODN into rat intact carotid artery
decreased p53 protein expression and resulted in a significant increase in neointimal lesion
growth at 2 and 4 weeks after balloon-angioplasty (Matsushita et al., 2000). Evidence suggests
that human cytomegalovirus infection contributes to the development of atherosclerosis and
restenosis, and part of this effect may be due to increased VSMC proliferation and migration by
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inactivation of p53 (Speir et al., 1994; Tanaka et al., 1999; Zhou et al., 1996; Zhou et al., 1999).
It is also noteworthy that human VSMCs from restenosis or in-stent stenosis sites demonstrate
normal or enhanced responses to p53 when compared to VSMCs from normal vessels (Scott et
al., 2002). Moreover, p53 gene transfer effectively inhibited neointimal hyperplasia after
experimental angioplasty (Matsushita et al., 2000; Scheinman et al., 1999; Yonemitsu et al.,
1998), and in human saphenous vein (George et al., 2001).
IID3. pRb
The complex interplay between pRb and transcription factors of the E2F family plays a
critical role in the control of cell growth (Stevaux and Dyson, 2002). E2F-dependent
transactivation of genes required for cell cycle progression is prevented in quiescent cells due to
the accumulation of hypophosphorylated pRb. Hyperphorylation of pRb by mitogenic stimuli
leads to E2F activation and cell growth. Transfer of antisense pRb ODN into human VSMCs
resulted in the induction of the proapoptotic factors bax and p53, and this was associated with
increased number of apoptotic cells and a higher rate of DNA synthesis (Aoki et al., 1999).
Inhibition of VSMC proliferation in vitro 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 (Chang et al., 1995b; Smith et al., 1997b). Similarly, adenoviral
transfer of the pRb related protein RB2/p130 inhibited VSMC proliferation in vitro and prevented
neointimal hyperplasia after experimental angioplasty (Claudio et al., 1999).
IID4. GATA-6
The GATA transcription factors play a critical role in the establishment of hematopoietic
cell lineages and during the development of the cardiovascular system (Simon, 1995). GATA-6 is
15
rapidly downregulated upon mitogen stimulation of quiescent VSMCs (Suzuki et al., 1996), and
overexpression of GATA-6 induced p21Cip1 expression and G1 cell cycle arrest (Perlman et al.,
1998). Importantly, p21Cip1-null mouse embryonic fibroblasts were refractory to the GATA-6induced growth inhibition (Perlman et al., 1998). The level of GATA-6 mRNA, protein, and
DNA-binding activity is transiently downregulated at early time points after balloon angioplasty
in the rat carotid artery, and reversal of GATA-6 downregulation by adenovirus-mediated
GATA-6 gene transfer to the vessel wall inhibited intimal hyperplasia in this animal model
(Mano et al., 1999).
IID5. GAX
Gax is a homeobox gene highly expressed in cultures of quiescent VSMCS, which is
rapidly downregulated in vitro upon growth factor stimulation of VSMCs, and after balloon
angioplasty in vivo (Gorski et al., 1993; Weir et al., 1995). Overexpression of GAX inhibited
VSMC proliferation in vitro and attenuated neointimal thickening in balloon-injured rat carotid
arteries in a p21Cip1-dependent manner (Perlman et al., 1999; Smith et al., 1997a). Percutaneous
delivery of the Gax gene also inhibited vessel stenosis in a rabbit model of balloon angioplasty
(Maillard et al., 1997).
IIE. Overexpression of transdominant negative mutants of positive cell cycle regulators.
IIE1. Ras
Ras-dependent signaling plays an important role in mitogen-stimulated cell growth (Pronk
and Bos, 1994). Ras is implicated in the activation of the G1 CDK/cyclin/E2F pathway (Aktas et
al., 1997; Kerkhoff and Rapp, 1997; Leone et al., 1997; Lloyd et al., 1997; Peeper et al., 1997;
Winston et al., 1996; Zou et al., 1997) and is critical for the normal induction of cyclin A
16
promoter activity and DNA synthesis in mitogen-stimulated VSMCs (Sylvester et al., 1998).
Consistent with these findings, local delivery of transdominant negative mutants of Ras
attenuated neointimal thickening after experimental balloon angioplasty (Indolfi et al., 1995;
Ueno et al., 1997b).
IIE2. Mitogen-activated protein kinases (MAPKs)
The MAPK pathway is critical in the transducction of proliferative signals in many
mammalian tissues, including the cardiovascular system (Bogoyevitch, 2000; Zou et al., 1998).
Several families of MAPKs have been described, including the stress-activated protein kinases/cjun NH2-terminal protein kinases (SAPKs/JNKs), extracellular signal-regulated kinases (ERKs),
and p38. JNKs and ERKs disclosed persistent hyperexpression and activation in atherosclerotic
lesions of cholesterol-fed rabbits, suggesting that these factors play critical roles in initiating and
perpetuating cell proliferation during the development of atherosclerosis (Hu et al., 2000;
Metzler et al., 2000). Likewise, angioplasty in porcine and rat arteries led to the rapid activation
of ERKs and JNKs (Koyama et al., 1998; Lai et al., 1996; Lille et al., 1997; Pyles et al., 1997).
Consistent with this notion, gene transfer of dominant-negative mutants of ERK or JNK
prevented neointimal formation in balloon-injured rat artery (Izumi et al., 2001).
III. Clinical studies
The antiproliferative approaches used so far for the treatment of cardiovascular disease
have focused on restenosis and graft atherosclerosis, during which neointimal hyperplasia is
rapid and localized. These disorders remain the major limitation of revascularization by
percutaneous transluminal angioplasty (PTCA) and artery bypass surgery.
IIIA. E2F ‘decoy’
17
Encouraging results of the E2F ‘decoy’ strategy in animal models of balloon angioplasty
and graft atherosclerosis (see above) led to the initiation of the first Project of Ex-vivo Vein graft
Engineering via Transfection (PREVENT I) trial (Mann et al., 1999). In this single-centre,
randomized, controlled gene therapy clinical trial, 41 patients undergoing bypass for the
treatment of peripheral arterial occlusions 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.
This procedure was associated with a 70-74% decrease in the level of PCNA and c-myc mRNA
expressed by the VSMCs in the 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 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 (Dzau et al., 2002).
IIIB. c-myc antisense ODN
Pharmacokinetics and clinical safety of ascending doses of c-myc antisense ODN (LR3280) administered after PTCA was assessed by Roque et al. (Roque et al., 2001a). Seventy
eight patients were randomized to receive either standard care (n = 26) or standard care and
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
decreasing 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
18
the basis for the evaluation of local delivery of c-myc antisense ODN for the prevention of
human vasculoproliferative disease.
Kutryk et al. recently reported the results of the Investigation by the Thoraxcenter of
Antisense DNA using Local delivery and IVUS after Coronary Stenting (ITALICS) trial (Kutryk
et al., 2002). This randomized, placebo controlled study was designed to determine the efficacy
of antisense ODN against c-myc in 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 cmyc as demonstrated by the analysis of 77 patients.
19
IV. CONCLUSIONS
Excessive cell proliferation within the arterial wall is thought to contribute to neointimal
thickening during the pathogenesis of atherosclerosis, in-stent restenosis, and vessel bypass graft
failure. Animal models of atherosclerosis have demonstrated an inverse correlation between
neointimal cell proliferation and atheroma size, suggesting that excessive cell growth prevails at
the onset of atherogenesis. Cell proliferation may also predominate at the early stages of human
atheroma development. Thus, given that patients frequently exhibit advanced atherosclerotic
plaques when first diagnosed, the potential benefit of antiproliferative strategies for the treatment
of human atherosclerosis is uncertain. The antiproliferative approaches used so far in the setting
of vascular obstructive disease have focused on restenosis and graft atherosclerosis, during
which neointimal hyperplasia is spatially localized and develops over a short period of time
(typically 2-12 months). Gene therapy is emerging as an attractive strategy in the treatment of
vascular proliferative disease due to minimally invasive and easily monitored gene delivery in
vascular interventions. Antiproliferative gene therapy strategies that have proven efficient in
inhibiting neointimal thickening in animal models of vascular obstructive disease include the use
of antisense- and ribozyme-mediated inactivation of positive cell cycle regulators,
overexpression of negative regulators of cell growth, and ‘decoy’ strategies to inactivate
transcription factors that promote cell cycle progression. Although some of these strategies have
shown encouraging results in humans, further studies are required to override the current
practical barriers and limitations placed on most clinical trials before gene therapy strategies
exhibit wide application in clinic. These should include the clarification of safety issues,
development of better gene delivery vectors, and improvement of transgene expression. Aside
from these technical improvements, significant effort in basic research is warranted to identify
more effective and safer treatment genes.
20
Acknowledgements
Work in the laboratory of V. Andrés is partially supported by the Ministerio de Ciencia y
Tecnología of Spain (MCyT) and Fondo Europeo de Desarrollo Regional (grants SAF2001-2358
and SAF2002-1143), and from Instituto de Salud Carlos III (ISCIII) (Red Centros C03/01). S.
M. Sanz and M. Gascón are predoctoral fellows of the ISCIII and MCyT, respectively.
21
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36
Table 1: Attenuation of neointimal thickening by antiproliferative gene therapy approaches in animal
models of vascular proliferative disease.
Strategy
Antisense (ODN)
Antisense (retrovirus)
Ribozyme
‘Decoy’ ODN
Oveexpression of
growth suppressors
Overexpression of
dominant-negative
mutants
Target gene
CDK2
CDC2
Cyclin B1
CDC2/PCNA
CDC2/PCNA
CDK2
c-myb *
c-myc *
c-myc *
PDGF receptor
Cyclin G1
PCNA
TGF-1
PDGF-A
12-lipoxygenase
E2F
E2F
AP-1
p21Cip1
p21Cip1
p27Kip1
pRb
RB2/p130
p53
GAX
GATA-6
RAS
ERK
JNK
Animal model
Balloon angioplasty (rat)
Balloon angioplasty (rat)
Balloon angioplasty (rat)
Graft arteriosclerosis (rabbit, rat)
Balloon angioplasty (rat)
Graft arteriosclerosis (mouse)
Balloon angioplasty (pig, rat)
Balloon angioplasty (rat, pig, rabbit)
Graft arteriosclerosis (pig)
Balloon angioplasty (rat)
Balloon angioplasty (rat)
Stent (pig)
Balloon angioplasty (rat)
Balloon angioplasty (rat)
Balloon angioplasty (rat)
Balloon angioplasty (rat, pig)
Graft arteriosclerosis (rabbit, mouse, monkey)
Balloon angioplasty (rat, rabbit, minipig)
Balloon angioplasty (rat, mouse, pig)
Graft arteriosclerosis (rabbit)
Balloon angioplasty (rat, pig)
Balloon angioplasty (rat, pig)
Balloon angioplasty (rat)
Balloon angioplasty (rabbit, rat)
Balloon angioplasty (rat, rabbit)
Balloon angioplasty (rat)
Balloon angioplasty (rat)
Balloon angioplasty (rat)
Balloon angioplasty (rat)
Ref.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
* These inhibitory effects might be caused by a nonantisense mechanism (Burgess et al., 1995;
Chavany et al., 1995; Guvakova et al., 1995; Villa et al., 1995; Wang et al., 1996).
References Table 1
1) Abe et al., 1994; Morishita et al., 1994a
2) Abe et al., 1994; Morishita et al., 1994b
3) Morishita et al., 1994b
4) Mann et al., 1995; Miniati et al., 2000
5) Morishita et al., 1993
6) Suzuki et al., 1997
7) Gunn et al., 1997; Simons et al., 1992
8) Bennett et al., 1994a; Kipshidze et al., 2001; Kipshidze et al., 2002; Shi et al., 1994b
9) Mannion et al., 1998
37
10) Cohen-Sacks et al., 2002
11) Zhu et al., 1997
12) Frimerman et al., 1999
13) Yamamoto et al., 2000
14) Kotani et al., 2003
15) Gu et al., 2001
16) Ahn et al., 2002a; Morishita et al., 1995; Nakamura et al., 2002
17) Ehsan et al., 2001; Kawauchi et al., 2000; Mann et al., 1997
18) Ahn et al., 2002b; Buchwald et al., 2002; Kume et al., 2002
19) Chang et al., 1995a; Condorelli et al., 2001; Ueno et al., 1997a; Yang et al., 1996
20) Bai et al., 1998
21) Chen et al., 1997; Tanner et al., 2000
22) Chang et al., 1995b; Smith et al., 1997b
23) Claudio et al., 1999
24) Matsushita et al., 2000; Scheinman et al., 1999; Yonemitsu et al., 1998
25) Maillard et al., 1997; Perlman et al., 1999; Smith et al., 1997a
26) Mano et al., 1999
27) Indolfi et al., 1995; Ueno et al., 1997b
28) Izumi et al., 2001
29) Izumi et al., 2001
38
Table 2: Gene therapy clinical trials for vascular proliferative disease based on cytostatic strategies.
Trial
PREVENT I
Outcome
70-74% decreases in
the level of positive
cell cycle regulators
expressed by VSMCs
in the vein, and
reduction in primary
graft failure
PREVENT II Randomized
E2F decoy ODN Autologous vein Larger patency and
multicenter,
ex vivo
graft failure after inhibition neointimal
double-blinded,
transfection
coronary artery
thickening
placebo-controlled of vein graft
bypass
ITALICS
Randomized,
c-myc antisense In-stent
No reduction in
placebo-controlled ODN delivery coronary
angiographic
after stent
restenosis
restenosis rate
implantation
PREVENT:
ITALICS:
Design
Randomized,
double-blinded,
single center
Strategy
E2F decoy ODN
ex vivo
transfection
of vein graft
Disease
Autologous
vein graft
failure after
peripheral
artery bypass
Refs.
Mann et
al., 1999
Dzau et al.,
2002
Kutryk et
al., 2002
Project of ex-vivo vein graft engineering via transfection
Investigation by the thoraxcenter of antisense DNA using local delivery and IVUS
after coronary stenting
39
FIGURE LEGENDS
Fig. 1. Neointimal lesion development in response to cardiovascular risk factors and
mechanical injury. Exposure of the arterial wall to cardiovascular risk factors and mechanical
injury leads to endothelial damage. Recruitment of circulating leukocytes is promoted by the
expression of adhesion molecules by the injured endothelial cells. Neointimal leukocites release
a plethora of cytokines and chemokines that initiate and perpetuate an inflammatory response,
which activates signal transduction pathways and transcription factors that promote the
hyperplastic growth of the lesion. Accumulation of noncellular material also contributes to
atheroma development.
Fig. 2. Early atherogenesis is associated with abundant cell proliferation within the
arterial wall. Immunohistochemical analysis of aortic arch cross-section of male New Zealand
rabbits fed control chow or a cholesterol-rich diet for 2 months. Animals were injected with 5bromodeoxyuridine (BrdU) prior to sacrifice. Specimens were incubated with anti-BrdU and
anti-RAM11 antibodies to monitor cell proliferation and to identify macrophages, respectively
(Cortés et al., 2002). Arrowheads point to the internal elastic lamina. Note lack of atherosclerosis
and undetectable immunoreactivity for BrdU and RAM11 within the aortic arch of control
rabbits. In contrast, prominent fatty streaks enriched in lipid-laden macrophages are seen in
cholesterol-fed animals. Some macrophages are also detected within the media. Abundant BrdU
immunoreactivity demonstrates a high proliferative activity, particularly within the
atherosclerotic lesion. All photomicrographs are at the same magnification.
Fig. 3. Control of mammalian cell cycle by CDK/cyclin holoenzyme and growth
suppresssors of the CKI family. Sequential activation of specific CDK/cyclin complexes leads
to progression through the different phases of the cell cycle. Inhibitory proteins of the CKI
family (Cip/Kip and Ink4) inhibit CDK/cyclin activity.
40
Fig. 4. Targeted gene inactivation by means of gene therapy strategies. Decoy
approach by delivering a double-stranded ODN corresponding to the optimum DNA recognition
sequence of the transcription factor of interest (TF) leads to attenuation of its interaction with the
authentic cis-elements in cellular target genes, thus resulting in reduced gene transcription.
Ribozymes inactivate the gene of interest by degrading their transcript. Antisense ODNs
hybridize in a complementary fashion and stoicheometrically with the target mRNA, thus
causing blockade of translation or synthesis of a truncated (inactive) protein.
41