Antiproliferative Strategies for the Treatment of Vascular Proliferative Disease
Vicente Andrés and Claudia Castro
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
1
ABSTRACT
Excessive cellular proliferation contributes to the pathobiology of vascular obstructive
disease (e. g., atherosclerosis, in-stent restenosis, transplant vasculopathy, and vessel bypass
graft failure). Therefore, anti-proliferative therapies may be a suitable approach in the treatment
of these disorders. Candidate targets for such strategies include the cyclin-dependent
kinase/cyclin holoenzymes, members of the cyclin-dependent kinase family of inhibitory
proteins, tumor suppressors, growth factors and transcription factors that control cell cycle
progression. In this review, we will discuss the use of pharmacological agents and gene therapy
approaches targeting cellular proliferation in animal models and clinical trials of cardiovascular
disease.
KEY WORDS: atherosclerosis, restenosis, bypass graft failure, cell cycle, pharmacological
therapy, gene therapy
LIST OF ABBREVIATIONS: apoE, apolipoprotein E; AP-1, activator protein-1; BMS, bare
metal stent; 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.
2
1. Introduction
2. Preclinical studies
2.1. Pharmacological therapies
2.1.1. CDK inhibitors
2.1.2. Rapamycin and everolimus
2.1.3. Microtubule polymerising agents (Paclitaxel, Docetaxel)
2.1.4. Tranilast
2.1.5. Troglitazone
2.2. Gene therapy
2.2.1. Antisense approach
2.2.1.1. CDKs and cyclins
2.2.1.2. Mitogen-responsive nuclear factors that promote cell growth
2.2.2. Ribozymes
2.2.3. Transcription factor ‘decoy’ strategies
2.2.3.1. E2F
2.2.3.2. AP-1
2.2.4. Overexpression of growth suppressors
2.2.4.1. CKIs
2.2.4.2. p53
2.2.4.3. pRb
2.2.4.4. GATA-6
2.2.4.5. GAX
2.2.5. Overexpression of transdominant negative mutants of positive cell cycle
regulators.
2.2.5.1. Ras
2.2.5.2. MAPKs
3. Clinical studies
3.1. Rapamycin
3.2. E2F ‘decoy’
3.3. Tranilast
3.4. Placlitaxel
3.5. c-myc antisense ODN
4. Concluding remarks
5. Acknowledegments
6. References
3
1. Introduction
Animal studies and 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 contributes
to neointimal lesion growth during atherogenesis [1,2]. Abundant proliferation of vascular cells
is an important component of the chronic inflammatory response associated to atherosclerosis
and related vascular occlusive diseases (e. g., in-stent restenosis, transplant vasculopathy, and
vessel bypass graft failure) [3,4]. Thus, understanding the molecular mechanisms that control
hyperplastic growth of vascular cells should help develop novel therapeutic strategies to reduce
neointimal thickening.
Although arterial cell proliferation occurs in animal models during all phases of
atherogenesis [1,5-7], studies with hyperlipidemic rabbits have shown an inverse correlation
between atheroma size and cellular proliferation within the atheromatous plaque [8-10].
Consistent with the response-to-injury hypothesis [1], medial cell proliferation at early stages of
atherogenesis in fat-fed rabbits increased as a function of intimal lesion size [5]. Experimental
angioplasty is also characterized by abundant proliferation of vascular smooth muscle cells
(VSMCs), followed by the reestablishment of the quiescent phenotype typically within 2-4
weeks [11-13]. 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 [14-26]. However, controversy exists regarding the magnitude
of the proliferative response, ranging from a very low index of cell proliferation
[15,16,18,20,22,26] to abundance of dividing cells [17,24,25,27]. Aside from methodological
issues (e. g., differences in the fixatives used for tissue preservation, antigen accessibility,
4
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 [28].
Proliferating cells within human atherosclerotic tissue include VSMCs, leukocytes and
endothelial cells (ECs) [14-16,18-20,22]. 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 [25]. Regarding the relative magnitude of intimal and medial cell
proliferation, analysis of human carotid plaques revealed more proliferative activity in the
intimal lesion versus the underlying media [20]. This study also disclosed differential
distribution of proliferating cells in the intima versus the media; while the prevailing
proliferative cell type in the intima was the monocyte/macrophage (46% versus 9.7% -actin
immunoreactive VSMCs, 14.3% ECs, 13.1% T lymphocytes), VSMCs were the preponderant
proliferating cell type in the media (44.4% versus 20% ECs, 13.0% monocyte/macrophages, and
14.3% T lymphocytes). It is noteworthy that studies in human peripheral and coronary lesions
have suggested more prominent proliferation in restenotic compared to primary lesions
[18,26,27]. Moreover, cultured VSMCs from human advanced primary stenosing disclosed
lower proliferative capacity than cells from fresh restenosing lesions [29]. Thus, similar to the
situation in animal models, 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 [30]. Mitogenic stimuli activate CDK/cyclin holoenzymes, thus
causing 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
5
and individual pocket proteins determines whether E2F proteins function as transcriptional
activators or repressors [31]. Interaction of CDK/cyclins with CDK inhibitory proteins (CKIs)
attenuates CDK activity and promotes growth arrest [32]. 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 Dassociated CDKs. Mitogenic and antimitogenic stimuli affect the rates of synthesis and
degradation of CKIs, as well as their redistribution among different CDK/cyclin pairs [32]. 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. Moreover, the protooncogen c-myc
plays a key role in p27 sequestration through modulation of the level of cyclin D and E proteins.
VSMC proliferation in the balloon-injured rat carotid artery is associated with a temporally
and spatially coordinated expression of CDKs and cyclins [23,33]. Moreover, induction of these
factors correlated with increased CDK2 and CDC2 activity [23,34], 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
[17,23,35], suggesting that induction of positive cell-cycle control genes is a hallmark of
vascular proliferative disease in human patients.
In the following sections, we will discuss the use of pharmacological agents (Table 1) and
gene therapy strategies targeting cellular proliferation in animal models (Table 2) and clinical
trials (Table 3) of cardiovascular disease.
2. Preclinical studies
2.1. Pharmacological therapies
2.1.1. CDK inhibitors
Over fifty low molecular weight pharmacological CDK inhibitors that target the ATP-
6
binding pocket of the catalytic site of CDKs have been identified and grouped in different
families (purines, alkaloids, indirubins, flavonoids, paullones, butyrolactone I, hymenialdisine
and pyrazolo[3,4-b]quinoxalines). Structural information on these agents and their application in
cancer therapy was recently reviewed [36,37]. The rat carotid model of balloon angioplasty has
been used to assess the therapeutical efficacy of some pharmacological CDK inhibitors. The
purine CVT-313 reduced neointimal lesion formation by 80% when delivered at a dose of 1.25
mg/kg for 15 minutes under pressure at the site of balloon angioplasty [38]. Likewise,
flavopiridol at 5 mg/kg administered orally for 5 days beginning at the day of balloon angioplasty
reduced neointima formation by 35% and by 39% at day 7 and 14 after intervention, respectively
[39].
2.1.2. Rapamycin and everolimus
Rapamycin (sirolimus) is a macrolide antibiotic with potent immunosuppressant,
antiproliferative and antimigartory properties [40]. The efficacy of rapamycin in attenuating
neointimal thickening by both alloimmune and mechanical injury has been demonstrated in
several animal models, including cardiac transplantation and restenosis post-angioplasty [41-48].
Although stabilization of the growth suppressor p27Kip1 appears to be a critical mediator of
rapamycin-dependent growth arrest in vitro [49,50], the in vivo therapeutic efficacy of rapamycin
in inhibiting neointima formation after mechanical injury was similar in wild-type and p27Kip1null mice [51]. Oral administration of everolimus (40-O-(2-hydroxyethyl)-rapamycin, also
dubbed RAD or certican) was well tolerated and suppressed in-stent neointimal growth in the
rabbit iliac artery [52]. Moreover, everolimus attenuated the development of macrotaline-induced
pulmonary vascular neointimal formation in pneumonectomized rats [53], and fluvastatin in
combination with everolimus significantly reduced graft vascular disease in rat cardiac allografts
[54].
7
2.1.3. Microtubule polymerising agents (Paclitaxel, Docetaxel)
Paclitaxel (Taxol) induces tubulin polymerization, thus stabilizing microtubules and
causing G2/M arrest [55]. Both continuous exposure to paclitaxel and applications for 24 hours
or even 20 minutes caused a complete and prolonged inhibition of the growth of human VSMCs
stimulated in culture with several mitogens, with an IC50 of 2.0 nmol/L [56]. Moreover, locally
delivered paclitaxel prevented neointimal thickening in animal models of balloon angioplasty
and arterial stent implantation [56-61], thus making this agent a promising candidate for local
antiproliferative therapy of restenosis. Of note, paclitaxel protected against vascular endothelial
growth factor-mediated increase in neointimal formation after balloon angioplasty in femoral
artery of cholesterol-fed rabbits [62]. Local delivery of docetaxel, a novel microtubule
polymerising agent, also reduced neointimal hyperplasia in a balloon-injured rabbit iliac artery
model [63].
2.1.4. Tranilast
The anti-inflammatory agent tranilast (SB 252218) attenuates mitogen-dependent
proliferation and migration of VSMCs [64,65]. These inhibitory effects of tranilast are associated
with increased expression of p53 and p21Cip1 and elevated complexing of p21Cip1 with CDK2 and
CDK4. Tranilast inhibited neointimal formation in the rat carotid model of balloon angioplasty
and in murine models of cardiac allograft vasculopathy, and these in vivo activities also
correlated with p21Cip1 upregulation [64,66-68]. Importantly, genetic inactivation of p21Cip1
abolished tranilast-dependent reduction of neointimal thickening in a murine model of
mechanical vascular injury [69], further implicating this CKI in the antiproliferative activity of
tranilast. Tranilast also attenuated the proinflammatory activity of human monocytes, adding to
its potential efficacy as a therapeutic agent in restenosis [70].
8
2.1.5. Troglitazone
The thiazolidinediones troglitazone and pioglitazone are novel insulin sensitizing agents
that have been shown to inhibit the mitogenic effect of several growth factors in cultured
VSMCs [71,72]. Inhibition of c-fos induction by troglitazone appeared to occur via a blockade of
the mitogen-activated protein kinase (MAPK) pathway. When examined in vivo, troglitazone
attenuated neointimal formation 14 days after balloon injury of the aorta compared with injured
rats that received no troglitazone [72].
2.2. Gene therapy
Antiproliferative gene therapy strategies designed for the treatment of experimental
cardiovascular disease can be grouped into two main categories: 1) Antisense approaches,
ribozymes, and transcription factor ‘decoy’ strategies to inactivate positive cell cycle regulators
(e. g., CDK/cyclins, protooncogenes, E2F, growth factors), and 2) Forced overexpression of
negative regulators of cell growth (e. g., CKIs, p53, pRb, GAX, and GATA-6).
2.2.1. Antisense approach
This strategy typically utilizes a synthetic antisense oligodeoxynucleotide (ODN) that
hybridizes in a complementary fashion and stoicheometrically with the target mRNA, thereby
inactivating the gene of interest.
2.2.1.1. CDKs and cyclins
Several ODN strategies targeting CDKs and cyclins have proven effective in reducing
neointimal lesion formation in animal models of balloon angioplasty, including ODN against
CDK2 [34,73], CDC2 [34,74], and cyclin B1 [74]. Interestingly, cotransfection of antisense
ODN against CDC2 kinase and cyclin B1 resulted in further inhibition of neointima formation,
9
as compared to blockade of either gene target alone [74]. 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 [75], whereas this approach had no effect in the coronary arteries of
pigs after balloon angioplasty [76]. Downregulation of cyclin G1 expression by retrovirusmediated antisense gene transfer inhibited VSMC proliferation and neointima formation after
balloon angioplasty [77]. ODN against CDK2 [78], and a combination of antisense ODN against
PCNA and CDC2 [79], also attenuated experimental graft atherosclerosis.
2.2.1.2. 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 activated in
serum-stimulated VSMCs, and their overexpression can induce VSMC proliferation in vitro [8087]. Higher levels of c-myc mRNA are present in VSMCs cultured from atheromatous plaques
than in VSMCs from normal arteries [88], and arterial injury induced the expression of several
“immediate-early” genes [89-92]. Antisense ODNs against c-myc and c-myb reportedly inhibited
in a sequence-specific manner both VSMC proliferation in vitro [83,93-100], and neointima
formation after angioplasty [98,100-104] and vein grafting [105] in vivo. However, other studies
have suggested that these inhibitory effects might be caused by a nonantisense mechanism [106110].
2.2.2. Ribozymes
Targeted gene inactivation can be achieved by the use of ribozymes, a unique class of
RNA molecules that catalytically cleave the specific target RNA. 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
10
human VSMCs, and efficiently inhibited the growth of these cells [111]. Likewise, cleavage of
the platelet-derived growth factor (PDGF) A-chain mRNA by hammerhead ribozyme attenuated
human and rat VSMC growth in vitro [112,113].
The first evidence that ribozymes might represent useful tools in cardiovascular therapy
came from studies using experimental models of angioplasty. Frimerman et al. reported the
efficacy of chimeric hammerhead ribozyme to PCNA in reducing stent-induced stenosis in a
porcine coronary model [114]. Moreover, ribozyme strategy against TGF-1 inhibited
neointimal formation after balloon injury in the rat carotid artery model [115].
2.2.3. Transcription factor ‘decoy’ strategies
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.
2.2.3.1. E2F
E2F participates in the transcriptional activation of several growth and cell-cycle regulators
(e. g., c-myc, pRb, cdc2, cyclin E, cyclin A), and genes encoding proteins that are required for
nucleotide and DNA biosynthesis (e. g., DNA polymerase , histone H2A, pcna, thymidine
kinase) [116,117]. E2F inactivation using synthetic ‘decoy’ ODN containing an E2F consensus
binding site prevented experimental neointimal thickening in balloon-injured arteries [118], vein
grafts [119,120], and cardiac allografts [121]. 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) [122]. CD-E2F 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.
11
2.2.3.2. 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 [8991,123,124]. AP-1 ‘decoy’ ODN delivery into cultured human VSMCs significantly reduced cell
number and TGF-1 production under conditions of PDGF stimulation [125], and attenuated
neointimal thickening when applied at the site of balloon angioplasty in rabbit carotid [125] and
minipig coronary arteries [126]. Compared to conventional phosphorothioated AP-1 decoy
ODN, circular dumbbell AP-1 ‘decoy’ ODN was more effective in inhibiting the proliferation of
VSMCs in vitro and neointimal hyperplasia in vivo [127].
2.2.4. Overexpression of growth suppressors
2.2.4.1. CKIs
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 [21,128]. The protective role of p27Kip1 against neointimal
thickening has been rigorously demonstrated in hypercholesterolemic apolipoprotein E (apoE)deficient mice, in which genetic inactivation of one or two p27Kip1 alleles progressively
accelerated atherogenesis [6]. However, neointimal hyperplasia after mechanical damage of the
arterial wall was similar in wild-type and p27Kip1-null mice [51]. Redundant roles between p21Cip1
and p27Kip1, or compensatory increase in p21Cip1 expression (or other CKIs) might account for the
lack of phenotype of p27Kip1-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
12
artery and saphenous vein, Yang et al. suggested that sustained p27Kip1 expression in spite of
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 [129]. Likewise, dissimilar
proliferative response of intimal and medial VSMCs towards basic fibroblast growth factor
(bFGF or FGF2) correlated with distinct expression of p15Ink4b and p27Kip1 in these cells [130].
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 [131].
Regarding the role of CKIs during human atherosclerosis, more frequent expression of
p27Kip1 and p21Cip1 has been reported within regions of human coronary atheromas not
undergoing proliferation [21]. Concordant expression of TGF- receptors I and II in virtually all
cells positive for p27Kip1 within human atherosclerotic plaques indicates that TGF-1 present in
these lesions may contribute to p27Kip1 upregulation [35]. 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 [132].
Ectopic expression of p21Cip1 and p27Kip1, but not p16Ink4a, significantly reduced neointimal
thickening in several animal models of angioplasty [128,133-137]. Overexpression of p21Cip1 also
attenuated neointimal lesion formation in a rabbit model of vein grafting [138].
2.2.4.2. p53
The transcription factor p53 functions as a tumor suppressor. p53 displays both
antiproliferative and proapoptotic actions that are likely to result from complex regulatory
networks, including 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.,
13
helicases and caspases). Transfection of antisense p53 ODN resulted in increased VSMC
proliferation in vitro [139,140], and VSMCs isolated from p53-deficient mice showed a higher
rate of proliferation and migration than wild-type counterparts [141]. Of note, mitogen-induced
downregulation of p53 in explanted porcine tunica media tissue preceded early migration and
proliferation of VSMCs [142]. Regarding the effects of p53 overexpression in cultured VSMCs,
Yonemitsu et al. reported G1 arrest after transduction with a p53 plasmidic expression vector
[143], while George et al. found no effect on VSMC proliferation but augmented apoptosis and
reduced migration upon adenovirus-mediated overexpression of p53 [144].
Genetic inactivation studies in hypercholesterolemic apoE and apoE*3-Leiden mice have
conclusively demonstrated a proatherogenic effect of p53 deficiency, although the relative
contribution of increased cellular proliferation and decreased apoptosis in these animal models
remains obscure [145,146]. Mice deficient for p53 also disclosed accelerated vein graft
atherosclerosis [141]. Regarding human atherosclerosis, p53 is overexpressed but not mutated in
human atherosclerotic tissue [147], 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 [132].
Animal and human studies suggest the potential contribution of p53 to the pathogenesis of
restenosis. 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 [140]. In humans, inactivation of p53 activity by cytomegalovirus
infection might increase the incidence of restenosis [148-151]. It is also noteworthy that human
VSMCs from restenosis or in-stent stenosis sites demonstrated normal or enhanced responses to
p53 when compared to VSMCs from normal vessels [152]. When taken together with the
observation that p53 gene transfer effectively inhibited neointimal hyperplasia after experimental
14
angioplasty [140,143,153], the above studies suggest that p53 overexpression might be a suitable
strategy in the treatment of clinical restenosis after revascularization.
2.2.4.3. pRb
The complex interplay between pRb and transcription factors of the E2F family plays a
critical role in the control of cell growth [31]. Quiescent cells accumulate hypophosphorylated
pRb, thus preventing E2F-dependent transactivation of genes required for cell cycle progression.
CDK-dependent hyperphosphorylation of pRb leads to E2F activation and thereby allows cell
growth. Transfer of antisense pRb ODN into human VSMCs resulted in the induction of p53 and
the proapoptotic factor bax, and this was associated with increased number of apoptotic cells and
a higher DNA synthesis rate [139]. Conversely, adenovirus-mediated transfer of full-length
constitutively active (nonphosphorylatable) and phosphorylation-competent pRb, or truncated
forms of pRb, inhibited VSMC proliferation in vitro and attenuated neointima formation after
balloon angioplasty [154,155]. Likewise, adenoviral transfer of the pRb related protein RB2/p130
inhibited VSMC proliferation in vitro and prevented neointimal hyperplasia after experimental
angioplasty [156].
2.2.4.4. 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 [157]. GATA-6 is rapidly
downregulated upon mitogen stimulation of quiescent VSMCs [158], and overexpression of
GATA-6 induced p21Cip1 expression and promoted VSMC growth arrest [159]. Importantly,
p21Cip1-null mouse embryonic fibroblasts were refractory to the GATA-6-induced growth
inhibition [159]. GATA-6 transcript, protein, and DNA-binding activity are transiently
downregulated at early time points after balloon angioplasty in rat carotid arteries, and reversal of
15
GATA-6 inactivation by adenovirus-mediated GATA-6 gene transfer to the vessel wall inhibited
lesion formation in this animal model [160].
2.2.4.5. GAX
The homeobox gene Gax is highly expressed in cultures of quiescent VSMCS, and its
mRNA is rapidly downregulated upon growth factor stimulation of VSMCs in vitro and after
balloon angioplasty in vivo [161,162]. Overexpression of GAX inhibited VSMC proliferation in
vitro and attenuated neointimal thickening in balloon-injured rat carotid arteries in a p21Cip1dependent manner [163,164]. Percutaneous delivery of the Gax gene also inhibited vessel
stenosis in a rabbit model of balloon angioplasty [165].
2.2.5. Overexpression of transdominant negative mutants of positive cell cycle regulators.
2.2.5.1. Ras
Ras-dependent signaling plays a critical role in mitogen-stimulated cell growth [166].
Evidence has been presented implicating Ras in the activation of the G1 CDK/cyclin/E2F
pathway [167-173]. Ras is critical for the normal induction of cyclin A promoter activity and
DNA synthesis in mitogen-stimulated VSMCs [91]. Consistent with these findings, local delivery
of transdominant negative mutants of Ras attenuated neointimal thickening after experimental
balloon angioplasty [174,175].
2.2.5.2. MAPKs
MAPKs play a critical role in transducing proliferative signals in many mammalian tissues,
including the cardiovascular system [176,177]. The MAPKs extracellular signal-regulated kinase
(ERK) and stress-activated protein kinases/c-jun NH2-terminal protein kinases (SAPKs/JNKs)
disclosed persistent hyperexpression and activation in atherosclerotic lesions of cholesterol-fed
16
rabbits, suggesting that these factors play critical roles in initiating and perpetuating cell
proliferation during the development of atherosclerosis [178,179]. Likewise, angioplasty in
porcine and rat arteries led to the rapid activation of ERKs and JNKs [180-183]. Consistent with
this notion, gene transfer of dominant-negative mutants of ERK or JNK prevented neointimal
formation in balloon-injured rat artery [184].
3. 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. In general, systemic
pharmacological approaches that proved effective in animal models have been unsuccessful in
reducing
the
incidence
of
clinical
restenosis,
including
antiplatelet,
anticoagulant,
antithrombotic, antiproliferative, antioxidant, and antiinflammatory agents [185-188]. The lack
of correlation between animal studies and clinical trials is likely the result of distinct vascular
responses to mechanical injury, and/or dissimilar pharmacokinetics in diverse animal species.
Owing that coronary stents represent almost 80% of the contemporary interventional procedures
for revascularization, local delivery of antiproliferative agents by means of drug releasing stents
is the focus of active research.
3.1. Rapamycin (sirolimus)
Sousa et al. assessed the safety and efficacy of sirolimus-coated stents in preventing
neointimal proliferation in coronary arteries of 30 patients with angina pectoris [189,190]. The
results of this small, noncontrolled registry trial demonstrated that implantation of sirolimuscoated stents is feasible and safe. Moreover, these authors reported minimal neointimal
17
proliferation at 4-, 6-, and 12-month follow-up, as determined by angiography or intravascular
ultrasound (IVUS) analysis. Likewise, Rensing et al. reported negligible neointimal hyperplasia
volume and percent obstruction volume by quantitative IVUS in a small-scale study with 15
patients treated with sirolimus-eluting coronary stents [191]. More recently, the results of the
Randomized Study With the Sirolimus-Eluting Bx Velocity Balloon-Expandable Stent (RAVEL)
trial have been reported [192-194]. This multicenter trial randomized 238 patients with de novo
lesions into sirolimus-eluting versus bare Bx velocity stents. Angiographic findings of this study
demonstrated prevention of neointimal proliferation and late lumen loss in patients receiving
sirolimus-eluting stents compared to the placebo group, without affecting the plaque burden
behind the struts and irrespective of the vessel size. The Sirolimus-Coated BX Velocity BalloonExpandable Stent in the Treatment of Patients with De Novo Coronary Artery Lesions (SIRIUS)
trial is an ongoing study conducted in 53 United States centers that randomized 1.100 patients
with de novo lesion into sirolimus-eluting and bare stents. Preliminary results also showed a
significant reduction in binary restenosis, late lumen loss and repeat revascularization rates
[195]. Collectively, these studies with rapamycin-eluting stents show considerable promise for
the prevention of restenosis as compared with standard coronary stents. The effectiveness of
rapamycin in preventing accelerated vasculopathy or chronic graft vascular disease after heart
transplantation is being evaluated in ongoing clinical trials.
3.2. E2F ‘decoy’
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) [196]. In this single-centre, randomized, controlled
gene therapy 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
18
scrambled-ODN-treated (n=8) human infrainguinal vein grafts. Ex vivo delivery of ODNs was
achieved intraoperatively via pressure-mediated transfection. Safe and effective means of ex vivo
transfection of harvested vein grafts was achieved with the E2F ‘decoy’ ODN, with 70-74%
decreases 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. This pilot
trial was followed by the ongoing PREVENT II protocol, a randomized, double-blinded, placebo
controlled Phase IIb trial with patients undergoing coronary artery bypass surgery. The results of
quantitative coronary angiography and IVUS showed larger patency and inhibition of neointimal
thickening in treated patients at 12 months after intervention [4].
3.3. Tranilast
The multicenter, randomized, double-blind, placebo-controlled, 255-patient Tranilast
REstenosis following Angioplasty Trial (TREAT-1) showed that oral administration of 600
mg/day of tranilast for 3 months markedly reduced the restenosis rate after PTCA for de novo
lesions, as determined by angiographic follow-up at 3 months. Likewise, the similarly designed
TREAT-2 trial demonstrated markedly reduced restenosis rate after PTCA by oral administration
of tranilast in both de novo and restenotic lesions by angiographic follow-up examination at 3
months [197]. However, results of the Prevention of REStenosis with Tranilast and its Outcomes
(PRESTO) trial have shown no improvement of the quantitative measures of restenosis or its
clinical sequelae at 9 months of follow up [198]. This double-blind, placebo-controlled trial was
designed to evaluate the effects of oral administration of tranilast on major adverse
cardiovascular events and to assess its efficacy in reducing clinical, angiographic, and IVUS
assessments of restenosis [198,199]. Primary end points (first occurrence of death, myocardial
infarction, or ischemia-driven target vessel revascularization within 9 months) were not different
between placebo and tranilast groups. Myocardial infarction was the only component of major
19
adverse cardiovascular events to show some evidence of a reduction with tranilast. In the followup angiographic substudy, minimal lumen diameter (MLD), as measured by quantitative
coronary angiography (n=2018 patients), and plaque volume, as measured by IVUS (n=1107
patients), were not different between the placebo and tranilast groups.
3.4. Paclitaxel
Liistro et al. evaluated the efficacy of QuaDS-QP2 stent coated with the paclitaxel
derivative 7-hexanoyltaxol (qDES stent) in fifteen consecutive patients with selective indication
to percutaneous coronary intervention for in-stent restenosis [200]. Minimal neointimal
hyperplasia was observed at the 6-month follow-up. However, the antiproliferative effect of the
qDES stent was not maintained at the 1-year follow-up, indicating that 7-hexanoyltaxol was
effective at delaying but not preventing the occurrence of angiographic restenosis. Kataoka et al.
reported recently the results of the Study to COmpare REstenosis rate between QueST and
QuaDS-QP2 (SCORE), a randomized, multicenter trial comparing the qDES 7-hexanoyltaxoleluting stents with bare metal stents (BMS) in the treatment of de novo coronary lesions [201].
Detailed IVUS analysis of 122 patients revealed comparable acute mechanical effects between
both groups. Compared to BMS, qDES stents showed reduced neointimal growth by 70% at the
tightest cross section and by 68% over the stented segment, resulting in a significantly larger
lumen. Likewise, Honda et al. reported minimal amount of neointimal proliferation in the stented
segment in 15 native coronary lesions treated with the qDES stent [202]. Although the long-term
benefits and limitations of this technology require further investigation, the reduction in
neointimal thickening demonstrated that local delivery of 7-hexanoyltaxol via eluting stents
might augment conventional mechanical treatment of atherosclerotic disease. Several ongoing
human trials are testing the efficacy of paclitaxel and derivatives in inhibiting restenosis when
locally delivered through drug releasing stents [4,55,203]. These include the TAXUS I
20
(paclitaxel, Boston Scientific NIRx trade mark stent), ASPECT Study (paclitaxel, Cook V-Flex
plus trade mark stent) and ELUTES, a multicenter, randomized, 180-patient pilot trial
investigating paclitaxel-coated Cook V-flex stent.
3.5. c-myc antisense ODN
Roque et al. assessed the clinical safety and pharmacokinetics of ascending doses of c-myc
antisense ODN (LR-3280) administered after PTCA [204]. Seventy eight patients were
randomized to receive either standard care (n = 26) or standard care and escalating doses of LR3280 (n = 52) (doses from 1 to 24 mg), administered into target vessel through a guiding
catheter. Pharmacokinetic data revealed that peak plasma concentrations of LR- 3280 occurred at
1 minute and rapidly decreased 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. recently reported the results of the Investigation by the Thoraxcenter of
Antisense DNA using Local delivery and IVUS after Coronary Stenting (ITALICS) trial [205].
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. Analysis of 77
patients demonstrated 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.
21
4. CONCLUDING REMARKS
Excessive cell proliferation is thought to contribute to neointimal thickening during the
pathogenesis of atherosclerosis, in-stent restenosis, transplant vasculopathy, and vessel bypass
graft failure. Thus, substantial efforts over the last years have led to the development of
antiproliferative strategies that efficiently limited these disorders in animal models. Oral
administration of several pharmacological agents that inhibited neontimal thickening in animal
models of vascular proliferative disease have generally failed in human trials. However,
promising results of clinical trials based on the use of drug eluting stents to locally deliver
antiproliferative agents (e. g., rapamycin, 7-hexanoyltaxol, paclitaxel) suggest that this approach
might find broad application to reduce the incidence of in-stent restenosis after surgical
revascularization. Owing that vascular interventions, both endovascular and open surgical, allow
minimally invasive, easily monitored gene delivery, gene therapy is emerging as an attractive
strategy in the treatment of vascular proliferative disease. Antiproliferative gene therapy
strategies 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. Despite some encouraging
results, further studies are required to override the current practical barriers and limitations
placed on most clinical trials before pharmacological and gene therapy strategies exhibit wide
application in clinic. These should include the clarification of safety issues, improved selectivity
of pharmacological inhibitors of cell growth, development of better gene delivery vectors and
delivery catheters, 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.
22
5. Acknowledgements
Work in the laboratory of V. Andrés is supported by the Spanish Ministerio de Ciencia y
Tecnología and Fondo Europeo de Desarrollo Regional (grants SAF2001-2358 and SAF20021143), and from Generalitat Valenciana (grants GV01-488 and CTGCA/2002/04). C. Castro is
the recipient of a fellowship from Agencia Española de Cooperación Internacional.
23
6. References
[1]. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med 1999; 340: 115-26.
[2]. Steinberg D. Atherogenesis in perspective: hypercholesterolemia and inflammation as
partners in crime. Nat Med 2002; 8: 1211-7.
[3]. Sartore S, Chiavegato A, Faggin E, Franch R, Puato M, Ausoni S, et al. Contribution
of adventitial fibroblasts to neointima formation and vascular remodeling: from innocent
bystander to active participant. Circ Res 2001; 89: 1111-21.
[4].
Dzau VJ, Braun-Dullaeus RC, Sedding DG. Vascular proliferation and
atherosclerosis: new perspectives and therapeutic strategies. Nat Med 2002; 8: 1249-56.
[5]. Cortés MJ, Díez-Juan A, Pérez P, Pérez-Roger I, Arroyo-Pellicer R, Andrés V.
Increased early atherogenesis in young versus old hypercholesterolemic rabbits by a mechanism
independent of arterial cell proliferation. FEBS Letters 2002; 522: 99-103.
[6]. Díez-Juan A, Andrés V. The growth suppressor p27Kip1 protects against diet-induced
atherosclerosis. FASEB J 2001; 15: 1989-95.
[7]. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993;
362: 801-9.
[8].
Spraragen SC, Bond VP, Dahl LK. Role of hyperplasia in vascular lesions of
cholesterol-fed rabbits studied with thymidine-3H autoradiography. Circ Res 1962; 11: 329-36.
[9]. McMillan GC, Stary HC. Preliminary experience with mitotic activity of cellular
elements in the atherosclerotic plaques of cholesterol-fed rabbits studied by labeling with
tritiated thymidine. Ann N Y Acad Sci 1968; 149: 699-709.
24
[10].
Rosenfeld ME, Ross R. Macrophage and smooth muscle cell proliferation in
atherosclerotic lesions of WHHL and comparably hypercholesterolemic fat-fed rabbits.
Arteriosclerosis 1990; 10: 680-7.
[11]. Bauters C, Isner JM. The biology of restenosis. Prog Cardiovasc Dis 1997; 40: 10716.
[12]. Andrés V. Control of vascular smooth muscle cell growth and its implication in
atherosclerosis and restenosis. Int J Molec Med 1998; 2: 81-9.
[13]. Libby P, Tanaka H. The molecular basis of restenosis. Prog Cardiovasc Dis 1997;
40: 97-106.
[14].
Burrig KF. The endothelium of advanced arteriosclerotic plaques in humans.
Arterioscler Thromb 1991; 11: 1678-89.
[15]. Gordon D, Reidy MA, Benditt EP, Schwartz SM. Cell proliferation in human
coronary arteries. Proc Natl Acad Sci U S A 1990; 87: 4600-4.
[16].
Katsuda S, Coltrera MD, Ross R, Gown AM. Human atherosclerosis. IV.
Immunocytochemical analysis of cell activation and proliferation in lesions of young adults. Am
J Pathol 1993; 142: 1787-93.
[17]. Kearney M, Pieczek A, Haley L, Losordo DW, Andrés V, Schainfield R, et al.
Histopathology of in-stent restenosis in patients with peripheral artery disease. Circulation 1997;
95: 1998-2002.
[18].
O'Brien ER, Alpers CE, Stewart DK, Ferguson M, Tran N, Gordon D, et al.
Proliferation in primary and restenotic coronary atherectomy tissue. Implications for
antiproliferative therapy. Circ Res 1993; 73: 223-31.
25
[19]. Orekhov AN, Andreeva ER, Mikhailova IA, Gordon D. Cell proliferation in normal
and atherosclerotic human aorta: proliferative splash in lipid-rich lesions. Atherosclerosis 1998;
139: 41-8.
[20].
Rekhter MD, Gordon D. Active proliferation of different cell types, including
lymphocytes, in human atherosclerotic plaques. Am J Pathol 1995; 147: 668-77.
[21]. Tanner FC, Yang Z-Y, Duckers E, Gordon D, Nabel GJ, Nabel EG. Expression of
cyclin-dependent kinase inhibitors in vascular disease. Circ Res 1998; 82: 396-403.
[22]. Veinot JP, Ma X, Jelley J, O'Brien ER. Preliminary clinical experience with the
pullback atherectomy catheter and the study of proliferation in coronary plaques. Can J Cardiol
1998; 14: 1457-63.
[23]. Wei GL, Krasinski K, Kearney M, Isner JM, Walsh K, Andrés V. Temporally and
spatially coordinated expression of cell cycle regulatory factors after angioplasty. Circ Res 1997;
80: 418-26.
[24]. Essed CE, Van den Brand M, Becker AE. Transluminal coronary angioplasty and
early restenosis. Fibrocellular occlusion after wall laceration. Br Heart J 1983; 49: 393-6.
[25]. Nobuyoshi M, Kimura T, Ohishi H, Horiuchi H, Nosaka H, Hamasaki N, et al.
Restenosis after percutaneous transluminal coronary angioplasty: pathologic observations in 20
patients. J Am Coll Cardiol 1991; 17: 433-9.
[26]. O'Brien ER, Urieli-Shoval S, Garvin MR, Stewart DK, Hinohara T, Simpson JB, et
al. Replication in restenotic atherectomy tissue. Atherosclerosis 2000; 152: 117-26.
26
[27]. Pickering JG, Weir L, Jekanowski J, Kearney MA, Isner JM. Proliferative activity in
peripheral and coronary atherosclerotic plaque among patients undergoing percutaneous
revascularization. J Clin Invest 1993; 91: 1469-80.
[28]. Isner JM. Vascular remodeling. Honey, I think I shrunk the artery. Circulation 1994;
89: 2937-41.
[29]. Dartsch PC, Voisard R, Bauriedel G, Hofling B, Betz E. Growth characteristics and
cytoskeletal organization of cultured smooth muscle cells from human primary stenosing and
restenosing lesions. Arteriosclerosis 1990; 10: 62-75.
[30]. Morgan DO. Principles of CDK regulation. Nature 1995; 374: 131-4.
[31]. Stevaux O, Dyson NJ. A revised picture of the E2F transcriptional network and RB
function. Curr Opin Cell Biol 2002; 14: 684-91.
[32].
Philipp-Staheli J, Payne SR, Kemp CJ. p27Kip1: regulation and function of a
haploinsufficient tumor suppressor and its misregulation in cancer. Exp Cell Res 2001; 264: 14868.
[33]. Braun-Dullaeus RC, Mann MJ, Seay U, Zhang L, von Der Leyen HE, Morris RE, et
al. Cell cycle protein expression in vascular smooth muscle cells in vitro and in vivo is regulated
through phosphatidylinositol 3-kinase and mammalian target of rapamycin. Arterioscler Thromb
Vasc Biol 2001; 21: 1152-8.
[34]. Abe J, Zhou W, Taguchi J, Takuwa N, Miki K, Okazaki H, et al. Suppression of
neointimal smooth muscle cell accumulation in vivo by antisense cdc2 and cdk2
oligonucleotides in rat carotid artery. Biochem Biophys Res Commun 1994; 198: 16-24.
27
[35].
Ihling C, Technau K, Gross V, Schulte-Monting J, Zeiher AM, Schaefer HE.
Concordant upregulation of type II-TGF-beta-receptor, the cyclin- dependent kinases inhibitor
p27Kip1 and cyclin E in human atherosclerotic tissue: implications for lesion cellularity.
Atherosclerosis 1999; 144: 7-14.
[36]. Fischer PM. Recent advances and new directions in the discovery and development
of cyclin-dependent kinase inhibitors. Curr Opin Drug Discov Dev 2001; 4: 623-34.
[37]. Ivorra C, Samyn H, Edo MD, Castro C, Sanz-González SM, Díez-Juan A, et al.
Inhibiting cyclin-dependent kinase/cyclin activity for the treatment of cancer and cardiovascular
disease. Curr Pharm Biotech 2003; In Press.
[38]. Brooks EE, Gray NS, Joly A, Kerwar SS, Lum R, Mackman RL, et al. CVT-313, a
specific and potent inhibitor of CDK2 that prevents neointimal proliferation. J Biol Chem 1997;
272: 29207-11.
[39]. Ruef J, Meshel AS, Hu Z, Horaist C, Ballinger CA, Thompson LJ, et al. Flavopiridol
inhibits smooth muscle cell proliferation in vitro and neointimal formation In vivo after carotid
injury in the rat. Circulation 1999; 100: 659-65.
[40]. Marx SO, Marks AR. Bench to bedside: the development of rapamycin and its
application to stent restenosis. Circulation 2001; 104: 852-5.
[41].
Meiser BM, Billingham ME, Morris RE. Effects of cyclosporin, FK506, and
rapamycin on graft-vessel disease. Lancet 1991; 338: 1297-8.
[42]. Gregory CR, Huie P, Billingham ME, Morris RE. Rapamycin inhibits arterial intimal
thickening caused by both alloimmune and mechanical injury. Its effect on cellular, growth
factor, and cytokine response in injured vessels. Transplantation 1993; 55: 1409-18.
28
[43]. Gregory CR, Huang X, Pratt RE, Dzau VJ, Shorthouse R, Billingham ME, et al.
Treatment with rapamycin and mycophenolic acid reduces arterial intimal thickening produced
by mechanical injury and allows endothelial replacement. Transplantation 1995; 59: 655-61.
[44]. Morris RE, Cao W, Huang X, Gregory CR, Billingham ME, Rowan R, et al.
Rapamycin (Sirolimus) inhibits vascular smooth muscle DNA synthesis in vitro and suppresses
narrowing in arterial allografts and in balloon- injured carotid arteries: evidence that rapamycin
antagonizes growth factor action on immune and nonimmune cells. Transplant Proc 1995; 27:
430-1.
[45]. Burke SE, Lubbers NL, Chen YW, Hsieh GC, Mollison KW, Luly JR, et al.
Neointimal formation after balloon-induced vascular injury in Yucatan minipigs is reduced by
oral rapamycin. J Cardiovasc Pharmacol 1999; 33: 829-35.
[46]. Gallo R, Padurean A, Jayaraman T, Marx S, Rogue M, Adelman S, et al. Inhibition
of intimal thickening after balloon angioplasty in porcine coronary arteries by targeting
regulators of the cell cycle. Circulation 1999; 99: 2164-70.
[47]. Poston RS, Billingham M, Hoyt EG, Pollard J, Shorthouse R, Morris RE, et al.
Rapamycin reverses chronic graft vascular disease in a novel cardiac allograft model. Circulation
1999; 100: 67-74.
[48]. Suzuki T, Kopia G, Hayashi S, Bailey LR, Llanos G, Wilensky R, et al. Stent-based
delivery of sirolimus reduces neointimal formation in a porcine coronary model. Circulation
2001; 104: 1188-93.
[49]. Nourse J, Firpo E, Flanagan WM, Coats S, Polyak K, Lee MH, et al. Interleukin-2mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin.
Nature 1994; 372: 570-3.
29
[50].
Luo Y, Marx SO, Kiyokawa H, Koff A, Massague J, Marks AR. Rapamycin
resistance tied to defective regulation of p27Kip1. Mol Cell Biol 1996; 16: 6744-51.
[51]. Roque M, Reis ED, Cordon-Cardo C, Taubman MB, Fallon JT, Fuster V, et al.
Effect of p27 deficiency and rapamycin on intimal hyperplasia: in vivo and in vitro studies using
a p27 knockout mouse model. Lab Invest 2001; 81: 895-903.
[52].
Farb A, John M, Acampado E, Kolodgie FD, Prescott MF, Virmani R. Oral
everolimus inhibits in-stent neointimal growth. Circulation 2002; 106: 2379-84.
[53]. Nishimura T, Faul JL, Berry GJ, Veve I, Pearl RG, Kao PN. 40-O-(2-hydroxyethyl)rapamycin attenuates pulmonary arterial hypertension and neointimal formation in rats. Am J
Respir Crit Care Med 2001; 163: 498-502.
[54]. Gregory CR, Katznelson S, Griffey SM, Kyles AE, Berryman ER. Fluvastatin in
combination with rad significantly reduces graft vascular disease in rat cardiac allografts.
Transplantation 2001; 72: 989-93.
[55].
Oberhoff M, Herdeg C, Baumbach A, Karsch KR. Stent-based antirestenotic
coatings (sirolimus/paclitaxel). Catheter Cardiovasc Interv 2002; 55: 404-8.
[56]. Axel DI, Kunrt W, Göggelmann C, Oberhoff M, Herdeg C, K üttner A, et al.
Paclitaxel inhibits arterial smooth muscle cell proliferation and migration in vitro and in vivo
using local drug delivery. Circulation 1997; 96: 636-45.
[57]. Sollott SJ, Cheng L, Pauly RR, Jenkins GM, Monticone RE, Kuzuya M, et al. Taxol
inhibits neointimal smooth muscle cell accumulation after angioplasty in the rat. J Clin Invest
1995; 95: 1869-76.
30
[58]. Farb A, Heller PF, Shroff S, Cheng L, Kolodgie FD, Carter AJ, et al. Pathological
analysis of local delivery of paclitaxel via a polymer- coated stent. Circulation 2001; 104: 473-9.
[59]. Oberhoff M, Herdeg C, Al Ghobainy R, Cetin S, Kuttner A, Horch B, et al. Local
delivery of paclitaxel using the double-balloon perfusion catheter before stenting in the porcine
coronary artery. Catheter Cardiovasc Interv 2001; 53: 562-8.
[60]. Oberhoff M, Kunert W, Herdeg C, Kuttner A, Kranzhofer A, Horch B, et al.
Inhibition of smooth muscle cell proliferation after local drug delivery of the antimitotic drug
paclitaxel using a porous balloon catheter. Basic Res Cardiol 2001; 96: 275-82.
[61]. Kolodgie FD, John M, Khurana C, Farb A, Wilson PS, Acampado E, et al. Sustained
reduction of in-stent neointimal growth with the use of a novel systemic nanoparticle paclitaxel.
Circulation 2002; 106: 1195-8.
[62]. Celletti FL, Waugh JM, Amabile PG, Kao EY, Boroumand S, Dake MD. Inhibition
of vascular endothelial growth factor-mediated neointima progression with angiostatin or
paclitaxel. J Vasc Interv Radiol 2002; 13: 703-7.
[63]. Yasuda S, Noguchi T, Gohda M, Arai T, Tsutsui N, Nakayama Y, et al. Local
delivery of low-dose docetaxel, a novel microtubule polymerizing agent, reduces neointimal
hyperplasia in a balloon-injured rabbit iliac artery model. Cardiovasc Res 2002; 53: 481-6.
[64]. Takahashi A, Taniguchi T, Ishikawa Y, Yokoyama M. Tranilast inhibits vascular
smooth muscle cell growth and intimal hyperplasia by induction of p21(waf1/cip1/sdi1) and p53.
Circ Res 1999; 84: 543-50.
31
[65]. Kusama H, Kikuchi S, Tazawa S, Katsuno K, Baba Y, Zhai YL, et al. Tranilast
inhibits the proliferation of human coronary smooth muscle cell through the activation of
p21waf1. Atherosclerosis 1999; 143: 307-13.
[66]. Ohlstein EH, Romanic AM, Clark LV, Kapadia RD, Sarkar SK, Gagnon R, et al.
Application of in vivo and ex vivo magnetic resonance imaging for evaluation of tranilast on
neointima formation following balloon angioplasty of the rat carotid artery. Cardiovasc Res
2000; 47: 759-68.
[67]. Izawa A, Suzuki J, Takahashi W, Amano J, Isobe M. Tranilast inhibits cardiac
allograft vasculopathy in association with p21(Waf1/Cip1) expression on neointimal cells in
murine cardiac transplantation model. Arterioscler Thromb Vasc Biol 2001; 21: 1172-8.
[68]. Saiura A, Sata M, Hirata Y, Nagai R, Makuuchi M. Tranilast inhibits transplantassociated coronary arteriosclerosis in a murine model of cardiac transplantation. Eur J
Pharmacol 2001; 433: 163-8.
[69]. Sata M, Takahashi A, Tanaka K, Washida M, Ishizaka N, Ako J, et al. Mouse
genetic evidence that tranilast reduces smooth muscle cell hyperplasia via a p21(WAF1)dependent pathway. Arterioscler Thromb Vasc Biol 2002; 22: 1305-9.
[70]. Capper EA, Roshak AK, Bolognese BJ, Podolin PL, Smith T, Dewitt DL, et al.
Modulation of human monocyte activities by tranilast, SB 252218, a compound demonstrating
efficacy in restenosis. J Pharmacol Exp Ther 2000; 295: 1061-9.
[71]. Dubey RK, Zhang HY, Reddy SR, Boegehold MA, Kotchen TA. Pioglitazone
attenuates hypertension and inhibits growth of renal arteriolar smooth muscle in rats. Am J
Physiol 1993; 265: R726-32.
32
[72]. Law RE, Meehan WP, Xi X-P, Graf K, Wuthrich DA, Coats W, et al. Troglitazone
inhibits vascular smooth muscle cell growth and intimal hyperplasia. J Clin Invest 1996; 98:
1897-905.
[73]. Morishita R, Gibbons GH, Ellison KE, Nakajima M, von der Leyen H, Zhang L, et
al. Intimal hyperplasia after vascular injury is inhibited by antisense cdk2 kinase
oligonucleotides. J Clin Invest 1994; 93: 1458-64.
[74]. Morishita R, Gibbons GH, Kaneda Y, Ogihara T, Dzau VJ. Pharmacokinetics of
antisense oligodeoxyribonucleotides (cyclin B1 and CDC 2 kinase) in the vessel wall in vivo:
enhanced therapeutic utility for restenosis by HVJ-liposome delivery. Gene 1994; 149: 13-9.
[75]. Morishita R, Gibbons GH, Ellison KE, Nakajima M, Zhang L, Kaneda Y, et al.
Single intraluminal delivery of antisense cdc2 kinase and proliferating-cell nuclear antigen
oligonucleotides results in chronic inhibition of neointimal hyperplasia. Proc Natl Acad Sci USA
1993; 90: 8474-8.
[76]. Robinson KA, Chronos NA, Schieffer E, Palmer SJ, Cipolla GD, Milner PG, et al.
Endoluminal local delivery of PCNA/cdc2 antisense oligonucleotides by porous balloon catheter
does not affect neointima formation or vessel size in the pig coronary artery model of
postangioplasty restenosis. Cathet Cardiovasc Diagn 1997; 41: 348-53.
[77].
Zhu NL, Wu L, Liu PX, Gordon EM, Anderson WF, Starnes VA, et al.
Downregulation of cyclin G1 expression by retrovirus-mediated antisense gene transfer inhibits
vascular smooth muscle cell proliferation and neointima formation. Circulation 1997; 96: 62835.
33
[78]. Suzuki J-I, Isobe M, Morishita R, Aoki M, Horie S, Okubo Y, et al. Prevention of
graft coronary arteriosclerosis by antisense cdk2 kinase oligonucleotide. Nat Med 1997; 3: 9003.
[79]. Mann M, Gibbons GH, Kernoff RS, Diet FP, Tsao PS, Cooke JP, et al. Genetic
engineering of vein grafts resistant to atherosclerosis. Proc Natl Acad Sci USA 1995; 92: 4502-6.
[80]. Castellot JJ, Cochran DL, Karnovsky MJ. Effect of heparin on vascular smooth
muscle cells. I. Cell metabolism. J Cell Physiol 1985; 124: 21-8.
[81]. Kindy MS, Sonenshein GE. Regulation of oncogene expression in cultured aortic
smooth muscle cells: post-transcriptional control of c-myc mRNA. J Biol Chem 1986; 261:
12865-8.
[82]. Reilly CF, Kindy MS, Brown KE, Rosenberg RD, Sonenshein GE. Heparin prevents
vascular smooth muscle cell progression through the G1 phase of the cell cycle. J Biol Chem
1989; 264: 6990-5.
[83]. Brown KE, Kindy MS, Sonenshein GE. Expression of the c-myb proto-oncogene in
bovine vascular smooth muscle cells. J Biol Chem 1992; 267: 4625-30.
[84]. Campan M, Desgranges C, Gadeau A-P, Millet D, Belloc F. Cell cycle dependent
gene expression in quiescent, stimulated, and asynchronously cycling arterial smooth muscle
cells in culture. J Cell Physiol 1992; 150: 493-500.
[85]. Bennett MR, Evan GI, Newby AC. Deregulated expression of the c-myc oncogene
abolishes inhibition of proliferation of rat vascular smooth muscle cells by serum reduction,
interferon-, heparin, and cyclic nucleotide analogues and induces apoptosis. Circ Res 1994; 74:
525-36.
34
[86]. Rothman A, Woler B, Button D, Taylor P. Immediate-early gene expression in
respose to hypertrophic and proliferative stimuli in pulmonary arterial smooth muscle cells. J
Biol Chem 1994; 269: 6399-404.
[87].
Gorski DH, Walsh K. Mitogen-responsive nuclear factors that mediate growth
control signals in vascular myocytes. Cardiovasc Res 1995; 30: 585-92.
[88]. Parkes JL, Cardell RR, Hubbard FC, Hubbard D, Meltzer A, Penn A. Cultured
human atherosclerotic plaque smooth muscle cells retain transforming potential and display
enhanced expression of the myc protooncogene. Am J Pathol 1991; 138: 765-75.
[89]. Miano JM, Tota RR, Vlasic N, Danishefsky KJ, Stemerman MB. Early protooncogene expression in rat aortic smooth muscle cells following endothelial removal. Am J
Pathol 1990; 137: 761-5.
[90]. Miano JM, Vlasic N, Tota RR, Stemerman MB. Localization of Fos and Jun proteins
in rat aortic smooth muscle cells after vascular injury. Am J Pathol 1993; 142: 715-24.
[91]. Sylvester AM, Chen D, Krasinski K, Andrés V. Role of c-fos and E2F in the
induction of cyclin A transcription and vascular smooth muscle cell proliferation. J Clin Invest
1998; 101: 940-8.
[92]. Lambert DL, Malik N, Shepherd L, Gunn J, Francis SE, King A, et al. Localization
of c-Myb and induction of apoptosis by antisense oligonucleotide c-Myb after angioplasty of
porcine coronary arteries. Arterioscler Thromb Vasc Biol 2001; 21: 1727-32.
[93]. Pukac LA, Castellot JJ, Jr., Wright TC, Jr., Caleb BL, Karnovsky MJ. Heparin
inhibits c-fos and c-myc mRNA expression in vascular smooth muscle cells. Cell Regul 1990; 1:
435-43.
35
[94]. Ebbecke M, Unterberg C, Buchwald A, Stohr S, Wiegand V. Antiproliferative
effects of a c-myc antisense oligonucleotide on human arterial smooth muscle cells. Basic Res
Cardiol 1992; 87: 585-91.
[95]. Simons M, Rosenberg RD. Antisense nonmuscle myosin heavy chain and c-myb
oligonucleotides supress smooth muscle cell proliferation in vitro. Circ Res 1992; 70: 835-43.
[96]. Shi Y, Hutchinson HG, Hall DJ, Zalewski A. Downregulation of c-myc expression
by antisense oligonucleotides inhibits proliferation of human smooth muscle cells. Circulation
1993; 88: 1190-5.
[97].
Biro S, Fu Y-M, Yu Z-X, Epstein SE. Inhibitory effects of antisense
oligodeoxynucleotides targeting c-myc mRNA on smooth muscle cell proliferation and
migration. Proc Natl Acad Sci U S A 1993; 90: 654-8.
[98]. Bennett MR, Anglin S, McEwan JR, Jagoe R, Newby AC, Evan GI. Inhibition of
vascular smooth muscle cell proliferation in vitro and in vivo by c-myc antisense
oligodeoxynucleotides. J Clin Invest 1994; 93: 820-8.
[99]. Shi Y, Dodge GR, Hall DJ, Desrochers PE, Fard A, Shaheen F, et al. Inhibition of
type 1 collagen synthesis in vascular smooth muscle cells by c-myc antisense oligomers.
Circulation 1994; 90: I-147.
[100]. Gunn J, Holt CM, Francis SE, Shepherd L, Grohmann M, Newman CM, et al. The
effect of oligonucleotides to c-myb on vascular smooth muscle cell proliferation and neointima
formation after porcine coronary angioplasty. Circ Res 1997; 80: 520-31.
36
[101]. Simons M, Edelman ER, DeKeyser J-L, Langer R, Rosenberg RD. Antisense c-myb
oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo. Nature 1992;
359: 67-70.
[102].
Shi Y, Fard A, Galeo A, Hutchinson HG, Vermani P, Dodge GR, et al.
Transcatheter delivery of c-myc antisense oligomers reduces neointimal formation in a porcine
model of coronary artery balloon injury. Circulation 1994; 90: 944-51.
[103]. Kipshidze N, Keane E, Stein D, Chawla P, Skrinska V, Shankar LR, et al. Local
delivery of c-myc neutrally charged antisense oligonucleotides with transport catheter inhibits
myointimal hyperplasia and positively affects vascular remodeling in the rabbit balloon injury
model. Catheter Cardiovasc Interv 2001; 54: 247-56.
[104].
Kipshidze NN, Kim HS, Iversen P, Yazdi HA, Bhargava B, New G, et al.
Intramural coronary delivery of advanced antisense oligonucleotides reduces neointimal
formation in the porcine stent restenosis model. J Am Coll Cardiol 2002; 39: 1686-91.
[105]. Mannion JD, Ormont ML, Magno MG, O'Brien JE, Shi Y, Zalewski A. Sustained
reduction of neointima with c-myc antisense oligonucleotides in saphenous vein grafts. Ann
Thorac Surg 1998; 66: 1948-52.
[106]. Burgess TL, Fisher EF, Ross SL, Bready JV, Qian YX, Bayewitch LA, et al. The
antiproliferative activity of c-myb and c-myc antisense oligonucleotides in smooth muscle cells
is caused by a nonantisense mechanism. Proc Natl Acad Sci U S A 1995; 92: 4051-5.
[107]. Wang W, Chen HJ, Schwartz A, Cannon PJ, Stein CA, Rabbani LE. Sequenceindependent inhibition of in vitro vascular smooth muscle cell proliferation, migration, and in
vivo neointimal formation by phosphorothioate oligodeoxynucleotides. J Clin Invest 1996; 98:
443-50.
37
[108]. Villa AE, Guzman LA, Poptic EJ, Labhasetwar V, D'Souza S, Farrell CL, et al.
Effects of antisense c-myb oligonucleotides on vascular smooth muscle cell proliferation and
response to vessel wall injury. Circ Res 1995; 76: 505-13.
[109].
Guvakova MA, Yakubov LA, Vlodavsky I, Tonkinson JL, Stein CA.
Phosphorothioate oligodeoxynucleotides bind to basic fibroblast growth factor, inhibit its
binding to cell surface receptors, and remove it from low affinity binding sites on extracellular
matrix. J Biol Chem 1995; 270: 2620-7.
[110]. Chavany C, Connell Y, Neckers L. Contribution of sequence and phosphorothioate
content to inhibition of cell growth and adhesion caused by c-myc antisense oligomers. Mol
Pharmacol 1995; 48: 738-46.
[111]. Su JZ, Fukuda N, Hu WY, Kanmatsuse K. Ribozyme to human TGF-beta1 mRNA
inhibits the proliferation of human vascular smooth muscle cells. Biochem Biophys Res
Commun 2000; 278: 401-7.
[112]. Hu WY, Fukuda N, Nakayama M, Kishioka H, Kanmatsuse K. Inhibition of
vascular smooth muscle cell proliferation by DNA-RNA chimeric hammerhead ribozyme
targeting to rat platelet-derived growth factor A-chain mRNA. J Hypertens 2001; 19: 203-12.
[113].
Hu WY, Fukuda N, Kishioka H, Nakayama M, Satoh C, Kanmatsuse K.
Hammerhead ribozyme targeting human platelet-derived growth factor A- chain mRNA
inhibited the proliferation of human vascular smooth muscle cells. Atherosclerosis 2001; 158:
321-9.
[114]. Frimerman A, Welch PJ, Jin X, Eigler N, Yei S, Forrester J, et al. Chimeric DNARNA hammerhead ribozyme to proliferating cell nuclear antigen reduces stent-induced stenosis
in a porcine coronary model. Circulation 1999; 99: 697-703.
38
[115]. Yamamoto K, Morishita R, Tomita N, Shimozato T, Nakagami H, Kikuchi A, et al.
Ribozyme oligonucleotides against transforming growth factor-beta inhibited neointimal
formation after vascular injury in rat model: potential application of ribozyme strategy to treat
cardiovascular disease. Circulation 2000; 102: 1308-14.
[116]. Dyson N. The regulation of E2F by pRB-family proteins. Genes Dev 1998; 12:
2245-62.
[117].
Lavia P, Jansen-Durr P. E2F target genes and cell-cycle checkpoint control.
Bioessays 1999; 21: 221-30.
[118]. Morishita R, Gibbons GH, Horiuchi M, Ellison KE, Nakajima M, Zhang L, et al. A
gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth
muscle cell proliferation in vivo. Proc Natl Acad Sci USA 1995; 92: 5855-9.
[119]. Mann MJ, Gibbons GH, Tsao PS, von der Leyen HE, Cooke JP, Buitrago R, et al.
Cell cycle inhibition preserves endothelial function in genetically engineered rabbit vein grafts. J
Clin Invest 1997; 99: 1295-301.
[120]. Ehsan A, Mann MJ, Dell'Acqua G, Dzau VJ. Long-term stabilization of vein graft
wall architecture and prolonged resistance to experimental atherosclerosis after E2F decoy
oligonucleotide gene therapy. J Thorac Cardiovasc Surg 2001; 121: 714-22.
[121]. Kawauchi M, Suzuki J, Morishita R, Wada Y, Izawa A, Tomita N, et al. Gene
therapy for attenuating cardiac allograft arteriopathy using ex vivo E2F decoy transfection by
HVJ-AVE-liposome method in mice and nonhuman primates. Circ Res 2000; 87: 1063-8.
39
[122]. Ahn JD, Morishita R, Kaneda Y, Kim HS, Chang YC, Lee KU, et al. Novel E2F
decoy oligodeoxynucleotides inhibit in vitro vascular smooth muscle cell proliferation and in
vivo neointimal hyperplasia. Gene Ther 2002; 9: 1682-92.
[123]. Hu Y, Cheng L, Hochleitner BW, Xu Q. Activation of mitogen-activated protein
kinases (ERK/JNK) and AP-1 transcription factor in rat carotid arteries after balloon injury.
Arterioscler Thromb Vasc Biol 1997; 17: 2808-16.
[124]. Andrés V, Ureña J, Poch E, Chen D, Goukassian D. The role of Sp1 in the
induction of p27 gene expression in vascular smooth muscle cells in vitro and after balloon
angioplasty. Arterioscl Thromb Vasc Biol 2001; 21: 342-7.
[125]. Kume M, Komori K, Matsumoto T, Onohara T, Takeuchi K, Yonemitsu Y, et al.
Administration of a decoy against the activator protein-1 binding site suppresses neointimal
thickening in rabbit balloon-injured arteries. Circulation 2002; 105: 1226-32.
[126]. Buchwald AB, Wagner AH, Webel C, Hecker M. Decoy oligodeoxynucleotide
against activator protein-1 reduces neointimal proliferation after coronary angioplasty in
hypercholesterolemic minipigs. J Am Coll Cardiol 2002; 39: 732-8.
[127]. Ahn JD, Morishita R, Kaneda Y, Lee SJ, Kwon KY, Choi SY, et al. Inhibitory
effects of novel AP-1 decoy oligodeoxynucleotides on vascular smooth muscle cell proliferation
in vitro and neointimal formation in vivo. Circ Res 2002; 90: 1325-32.
[128].
Chen D, Krasinski K, Chen D, Sylvester A, Chen J, Nisen PD, et al.
Downregulation of cyclin-dependent kinase 2 activity and cyclin A promoter activity in vascular
smooth muscle cells by p27Kip1, an inhibitor of neointima formation in the rat carotid artery. J
Clin Invest 1997; 99: 2334-41.
40
[129].
Yang Z, Oemar BS, Carrel T, Kipfer B, Julmy F, Lüscher TF. Different
proliferative properties of smooth muscle cells of human arterial and venous bypass vessels: role
of PDGF receptors, mitogen-activated protein kinase, and cyclin-dependent kinase inhibitors.
Circulation 1998; 97: 181-7.
[130]. Olson NE, Kozlowski J, Reidy MA. Proliferation of intimal smooth muscle cells.
Attenuation of basic fibroblast growth factor 2-stimulated proliferation is associated with
increased expression of cell cycle inhibitors. J Biol Chem 2000; 275: 11270-7.
[131]. Castro C, Díez-Juan A, Cortés MJ, Andrés V. Distinct regulation of mitogenactivated protein kinases and p27Kip1 in smooth muscle cells from different vascular beds: A
potential role in establishing regional phenotypic variance. J Biol Chem 2003; In Press.
[132].
Ihling C, Menzel G, Wellens E, Monting JS, Schaefer HE, Zeiher AM.
Topographical association between the cyclin-dependent kinases inhibitor P21, p53
accumulation, and cellular proliferation in human atherosclerotic tissue. Arterioscler Thromb
Vasc Biol 1997; 17: 2218-24.
[133]. Chang MW, Barr E, Lu MM, Barton K, Leiden JM. Adenovirus-mediated overexpression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle
cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty.
J Clin Invest 1995; 96: 2260-8.
[134]. Ueno H, Masuda S, SNishio S, Li JJ, Yamamoto H, Takeshita A. Adenovirusmediated transfer of cyclin-dependent kinase inhibitor p21 suppresses neointimal formation in
the balloon-injured rat carotid arteries in vivo. Ann N Y Acad Sci 1997; 811: 401-11.
41
[135]. Yang Z-Y, Simari RD, Perkins ND, San H, Gordon D, Nabel GJ, et al. Role of p21
cyclin-dependent kinase inhibitor in limiting intimal cell proliferation in response to arterial
injury. Proc Natl Acad Sci USA 1996; 93: 7905-10.
[136]. Condorelli G, Aycock JK, Frati G, Napoli C. Mutated p21/WAF/CIP transgene
overexpression reduces smooth muscle cell proliferation, macrophage deposition, oxidationsensitive mechanisms, and restenosis in hypercholesterolemic apolipoprotein E knockout mice.
FASEB J 2001; 15: 2162-70.
[137]. Tanner FC, Boehm M, Akyürek LM, San H, Yang Z-Y, Tashiro J, et al. Differential
effects of the cyclin-dependent kinase inhibitors p27Kip1, p21Cip1, and p16Ink4 on vascular smooth
muscle cell proliferation. Circulation 2000; 101: 2022-5.
[138]. Bai H, Morishita R, Kida I, Yamakawa T, Zhang W, Aoki M, et al. Inhibition of
intimal hyperplasia after vein grafting by in vivo transfer of human senescent cell-derived
inhibitor-1 gene. Gene Ther 1998; 5: 761-9.
[139]. Aoki M, Morishita R, Matsushita H, Hayashi S, Nakagami H, Yamamoto K, et al.
Inhibition of the p53 tumor suppressor gene results in growth of human aortic vascular smooth
muscle cells. Potential role of p53 in regulation of vascular smooth muscle cell growth.
Hypertension 1999; 34: 192-200.
[140]. Matsushita H, Morishita R, Aoki M, Tomita N, Taniyama Y, Nakagami H, et al.
Transfection of antisense p53 tumor suppressor gene oligodeoxynucleotides into rat carotid
artery results in abnormal growth of vascular smooth muscle cells. Circulation 2000; 101: 144752.
[141]. Mayr U, Mayr M, Li C, Wernig F, Dietrich H, Hu Y, et al. Loss of p53 accelerates
neointimal lesions of vein bypass grafts in mice. Circ Res 2002; 90: 197-204.
42
[142]. Rodriguez-Campos A, Ruiz-Enriquez P, Faraudo S, Badimon L. Mitogen-induced
p53 downregulation precedes vascular smooth muscle cell migration from healthy tunica media
and proliferation. Arterioscler Thromb Vasc Biol 2001; 21: 214-9.
[143]. Yonemitsu Y, Kaneda Y, Tanaka S, Nakashima Y, Komori K, Sugimachi K, et al.
Transfer of wild-type p53 gene effectively inhibits vascular smooth muscle cell proliferation in
vitro and in vivo. Circ Res 1998; 82: 147-56.
[144]. George SJ, Angelini GD, Capogrossi MC, Baker AH. Wild-type p53 gene transfer
inhibits neointima formation in human saphenous vein by modulation of smooth muscle cell
migration and induction of apoptosis. Gene Ther 2001; 8: 668-76.
[145]. Guevara NV, Kim HS, Antonova EI, Chan L. The absence of p53 accelerates
atherosclerosis by increasing cell proliferation in vivo. Nat Med 1999; 5: 335-9.
[146]. van Vlijmen BJ, Gerritsen G, Franken AL, Boesten LS, Kockx MM, Gijbels MJ, et
al. Macrophage p53 deficiency leads to enhanced atherosclerosis in APOE*3- Leiden transgenic
mice. Circ Res 2001; 88: 780-6.
[147]. Iacopetta B, Wysocki S, Norman P, House A. The p53 tumor suppressor gene is
overexpressed but not mutated in human atherosclerotic tissue. Int J Oncol 1995; 7: 399-402.
[148]. Speir E, Modali R, Huang E-S. Potential role of human cytomegalovirus and p53
interaction in coronary restenosis. Science 1994; 265: 391-4.
[149]. Zhou YF, Leon MB, Waclawiw MA, Popma JJ, Yu ZX, Finkel T, et al. Association
between prior cytomegalovirus infection and the risk of restenosis after coronary atherectomy. N
Engl J Med 1996; 335: 624-30.
43
[150]. Zhou YF, Yu ZX, Wanishsawad C, Shou M, Epstein SE. The immediate early gene
products of human cytomegalovirus increase vascular smooth muscle cell migration,
proliferation, and expression of PDGF beta-receptor. Biochem Biophys Res Commun 1999; 256:
608-13.
[151]. Tanaka K, Zou JP, Takeda K, Ferrans VJ, Sandford GR, Johnson TM, et al. Effects
of human cytomegalovirus immediate-early proteins on p53-mediated apoptosis in coronary
artery smooth muscle cells. Circulation 1999; 99: 1656-9.
[152]. Scott S, O'Sullivan M, Hafizi S, Shapiro LM, Bennett MR. Human vascular smooth
muscle cells from restenosis or in-stent stenosis sites demonstrate enhanced responses to p53.
Implications for brachytherapy and drug treatment for restenosis. Circ Res 2002; 90: 398-404.
[153]. Scheinman M, Ascher E, Levi GS, Hingorani A, Shirazian D, Seth P. p53 gene
transfer to the injured rat carotid artery decreases neointimal formation. J Vasc Surg 1999; 29:
360-9.
[154]. Chang MW, Barr E, Seltzer J, Jiang Y, Nabel GJ, Nabel EG, et al. Cytostatic gene
therapy for vascular proliferative disorders with a constitutively active form of the
retinoblastoma gene product. Science 1995; 267: 518-22.
[155]. Smith RC, Wills KN, Antelman D, Perlman H, Truong LN, Krasinski K, et al.
Adenoviral constructs encoding phosphorylation-competent full-length and truncated forms of
the human retinoblastoma protein inhibit myocyte proliferation and neointima formation.
Circulation 1997; 96: 1899-905.
[156]. Claudio PP, Fratta L, Farina F, Howard CM, Stassi G, Numata S, et al. Adenoviral
RB2/p130 gene transfer inhibits smooth muscle cell proliferation and prevents restenosis after
angioplasty. Circ Res 1999; 85: 1032-9.
44
[157]. Simon MC. Gotta have GATA. Nat Genetics 1995; 11: 9-11.
[158]. Suzuki E, Evans T, Lowry J, Truong L, Bell DW, Testa JR, et al. The human
GATA-6 gene: structure, chromosomal location and regulation of expression by tissue-specific
and mitogen-responsive signals. Genomics 1996; 38: 283-90.
[159]. Perlman H, Suzuki E, Simonson M, Smith RC, Walsh K. GATA-6 induces p21Cip1
expression and G1 cell cycle arrest. J Biol Chem 1998; 273: 13713-8.
[160]. Mano T, Luo ZY, Malendowicz SL, Evans T, Walsh K. Reversal of GATA-6
downregulation promotes smooth muscle differentiation and inhibits intimal hyperplasia in
balloon-injured rat carotid artery. Circ Res 1999; 84: 647-54.
[161]. Gorski DH, LePage DF, Patel CV, Copeland NG, Jenkins NA, Walsh K. Molecular
cloning of a diverged homeobox gene that is rapidly down-regulated during the G0/G1 transition
in vascular smooth muscle cells. Mol Cell Biol 1993; 13: 3722-33.
[162]. Weir L, Chen D, Pastore C, Isner JM, Walsh K. Expression of GAX, a growtharrest homeobox gene, is rapidly down-regulated in the rat carotid artery during the proliferative
response to balloon injury. J Biol Chem 1995; 270: 5457-61.
[163]. Smith RC, Branellec D, Gorski DH, Guo K, Perlman H, Dedieu J-F, et al. p21CIP1mediated inhibition of cell proliferation by overexpression of the Gax homeodomain gene. Genes
Dev 1997; 11: 1674-89.
[164]. Perlman H, Luo Z, Krasinski K, Le Roux A, Mahfoudi A, Smith RC, et al.
Adenovirus-mediated delivery of the Gax transcription factor to rat carotid arteries inhibits
smooth muscle proliferation and induces apoptosis. Gene Ther 1999; 6: 758-63.
45
[165].
Maillard L, Van Belle E, Smith RC, Le Roux A, Denèfle P, Steg G, et al.
Percutaneous delivery of the gax gene inhibits vessel stenosis in a rabbit model of balloon
angioplasty. Cardiovasc Res 1997; 35: 536-46.
[166]. Pronk GJ, Bos JL. The role of p21ras in receptor tyrosine kinase signalling.
Biochim Biophys Acta 1994; 1198: 131-47.
[167]. Winston JT, Coats SR, Wang Y-Z, Pledger WJ. Regulation of the cell cycle
machinery by oncogenic ras. Oncogene 1996; 12: 127-34.
[168]. Aktas H, Cai H, Cooper GM. Ras links growth factor signaling to the cell cyle
machinery via regulation of cyclin D1 and the cdk inhibitor p27KIP1. Mol Cell Biol 1997; 17:
3850-7.
[169]. Kerkhoff E, Rapp UR. Induction of cell proliferation in quiescent NIH 3T3 cells by
oncogenic c-Raf-1. Mol Cell Biol 1997; 17: 2576-86.
[170]. Leone G, DeGregori J, Sears R, Jakoi L, Nevins JR. Myc and Ras collaborate in
inducing accumulation of active cyclin E/cdk2 and E2F. Nature 1997; 387: 422-6.
[171]. Lloyd AC, Obermüller F, Staddon S, Barth CF, McMahon M, Land H. Cooperating
oncogenes converge to regulate cyclin/cdk complexes. Genes Dev 1997; 11: 663-77.
[172]. Peeper DS, Upton TM, Ladha MH, Neuman E, Zalvide J, Bernards R, et al. Ras
signalling linked to the cell-cycle machinery by the retinoblastoma protein. Nature 1997; 386:
177-81.
[173]. Zou X, Rudchenko S, Wong K-k, Calame K. Induction of c-myc transcription by
the v-Abl tyrosine kinase requires Ras, Raf1, and cyclin-dependent kinases. Genes Dev 1997;
11: 654-62.
46
[174]. Indolfi C, Avvedimento EV, Rapacciuolo A, Di Lorenzo E, Esposito G, Stabile E,
et al. Inhibition of cellular ras prevents smooth muscle cell proliferation after vascular injury in
vivo. Nature Med 1995; 1: 541-5.
[175]. Ueno H, Yamamoto H, Ito S-i, Li J-J, Takeshita A. Adenovirus-mediated transfer
of a dominant-negative H-ras suppresses neointimal formation in balloon-injured arteries in
vivo. Arterioscler Thromb Vasc Biol 1997; 17: 898-904.
[176]. Bogoyevitch MA. Signalling via stress-activated mitogen-activated protein kinases
in the cardiovascular system. Cardiovasc Res 2000; 45: 826-42.
[177]. Zou Y, Hu Y, Metzler B, Xu Q. Signal transduction in arteriosclerosis: mechanical
stress-activated MAP kinases in vascular smooth muscle cells (review). Int J Mol Med 1998; 1:
827-34.
[178]. Metzler B, Hu Y, Dietrich H, Xu Q. Increased expression and activation of stressactivated protein kinases/c-Jun NH(2)-terminal protein kinases in atherosclerotic lesions coincide
with p53. Am J Pathol 2000; 156: 1875-86.
[179]. Hu Y, Dietrich H, Metzler B, Wick G, Xu Q. Hyperexpression and activation of
extracellular signal-regulated kinases (ERK1/2) in atherosclerotic lesions of cholesterol-fed
rabbits. Arterioscler Thromb Vasc Biol 2000; 20: 18-26.
[180]. Lai K, Lee W-S, Jain MK, Lee M-E, Haber E. Mitogen-activated protein kinase
phosphatase-1 in rat arterial smooth muscle cell proliferation. J Clin Invest 1996; 98: 1560-7.
[181]. Lille S, Daum G, Clowes MM, Clowes AW. The regulation of p42/p44 mitogenactivated protein kinases in the injured rat carotid artery. J Surg Res 1997; 70: 178-86.
47
[182]. Pyles JM, March KL, Mehdi K, Wilenski RL, Adam LP. Activation of MAP
Kinase in vivo follows balloon overstretch injury of porcine coronary and carotid arteries. Circ
Res 1997; 81: 904-10.
[183]. Koyama H, Olson NE, Dastvan FF, Reidy MA. Cell replication in the arterial wall:
activation of signaling pathway following in vivo injury. Circ Res 1998; 82: 713-21.
[184]. Izumi Y, Kim S, Namba M, Yasumoto H, Miyazaki H, Hoshiga M, et al. Gene
transfer of dominant-negative mutants of extracellular signal- regulated kinase and c-Jun NH2terminal kinase prevents neointimal formation in balloon-injured rat artery. Circ Res 2001; 88:
1120-6.
[185]. Califf RM, Fortin DF, Frid DJ, Harlan WR, Ohman EM, Bengtson JR, et al.
Restenosis after coronary angioplasty: an overview. J Am Coll Cardiol 1991; 17: 2B-13B.
[186]. Chan AW, Chew DP, Lincoff AM. Update on Pharmacology for Restenosis. Curr
Interv Cardiol Rep 2001; 3: 149-55.
[187]. Franklin SM, Faxon DP. Pharmacologic prevention of restenosis after coronary
angioplasty: review of the randomized clinical trials. Coronary Artery Dis 1993; 4: 232-42.
[188].
Popma JJ, Califf RM, Topol EJ. Clinical trials of restenosis after coronary
angioplasty. Circulation 1991; 84: 1426-36.
[189]. Sousa JE, Costa MA, Abizaid A, Abizaid AS, Feres F, Pinto IM, et al. Lack of
neointimal proliferation after implantation of sirolimus-coated stents in human coronary arteries:
a quantitative coronary angiography and three-dimensional intravascular ultrasound study.
Circulation 2001; 103: 192-5.
48
[190]. Sousa JE, Costa MA, Abizaid AC, Rensing BJ, Abizaid AS, Tanajura LF, et al.
Sustained suppression of neointimal proliferation by sirolimus-eluting stents: one-year
angiographic and intravascular ultrasound follow-up. Circulation 2001; 104: 2007-11.
[191]. Rensing BJ, Vos J, Smits PC, Foley DP, van den Brand MJ, van der Giessen WJ, et
al. Coronary restenosis elimination with a sirolimus eluting stent: first European human
experience with 6-month angiographic and intravascular ultrasonic follow-up. Eur Heart J 2001;
22: 2125-30.
[192]. Regar E, Serruys PW, Bode C, Holubarsch C, Guermonprez JL, Wijns W, et al.
Angiographic findings of the multicenter Randomized Study With the Sirolimus-Eluting Bx
Velocity Balloon-Expandable Stent (RAVEL): sirolimus-eluting stents inhibit restenosis
irrespective of the vessel size. Circulation 2002; 106: 1949-56.
[193]. Serruys PW, Degertekin M, Tanabe K, Abizaid A, Sousa JE, Colombo A, et al.
Intravascular ultrasound findings in the multicenter, randomized, double-blind RAVEL
(RAndomized study with the sirolimus-eluting VElocity balloon-expandable stent in the
treatment of patients with de novo native coronary artery Lesions) trial. Circulation 2002; 106:
798-803.
[194]. Morice MC, Serruys PW, Sousa JE, Fajadet J, Ban Hayashi E, Perin M, et al. A
randomized comparison of a sirolimus-eluting stent with a standard stent for coronary
revascularization. N Engl J Med 2002; 346: 1773-80.
[195]. Degertekin M, Regar E, Tanabe K, Lee CH, Serruys PW. Sirolimus eluting stent in
the treatment of atherosclerosis coronary artery disease. Minerva Cardioangiol 2002; 50: 405-18.
49
[196]. Mann MJ, Whittemore AD, Donaldson MC, Belkin M, Conte MS, Polak JF, et al.
Ex-vivo gene therapy of human vascular bypass grafts with E2F decoy: the PREVENT singlecentre, randomised, controlled trial. Lancet 1999; 354: 1493-8.
[197]. Tamai H, Katoh K, Yamaguchi T, Hayakawa H, Kanmatsuse K, Haze K, et al. The
impact of tranilast on restenosis after coronary angioplasty: the Second Tranilast Restenosis
Following Angioplasty Trial (TREAT-2). Am Heart J 2002; 143: 506-13.
[198]. Holmes DR, Jr., Savage M, LaBlanche JM, Grip L, Serruys PW, Fitzgerald P, et al.
Results of Prevention of REStenosis with Tranilast and its Outcomes (PRESTO) trial.
Circulation 2002; 106: 1243-50.
[199]. Holmes D, Fitzgerald P, Goldberg S, LaBlanche J, Lincoff AM, Savage M, et al.
The PRESTO (Prevention of restenosis with tranilast and its outcomes) protocol: a double-blind,
placebo-controlled trial. Am Heart J 2000; 139: 23-31.
[200]. Liistro F, Stankovic G, Di Mario C, Takagi T, Chieffo A, Moshiri S, et al. First
clinical experience with a paclitaxel derivate-eluting polymer stent system implantation for instent restenosis: immediate and long- term clinical and angiographic outcome. Circulation 2002;
105: 1883-6.
[201].
Kataoka T, Grube E, Honda Y, Morino Y, Hur SH, Bonneau HN, et al. 7-
hexanoyltaxol-eluting stent for prevention of neointimal growth: an intravascular ultrasound
analysis from the Study to COmpare REstenosis rate between QueST and QuaDS-QP2
(SCORE). Circulation 2002; 106: 1788-93.
[202]. Honda Y, Grube E, de La Fuente LM, Yock PG, Stertzer SH, Fitzgerald PJ. Novel
drug-delivery stent: intravascular ultrasound observations from the first human experience with
the QP2-eluting polymer stent system. Circulation 2001; 104: 380-3.
50
[203]. Muller R, Bullesfeld L, Gerckens U, Grube E. State of treatment of coronary artery
disease by drug releasing stents. Herz 2002; 27: 508-13.
[204]. Roque F, Mon G, Belardi J, Rodriguez A, Grinfeld L, Long R, et al. Safety of
intracoronary administration of c-myc antisense oligomers after percutaneous transluminal
coronary angioplasty (PTCA). Antisense Nucleic Acid Drug Dev 2001; 11: 99-106.
[205]. Kutryk MJ, Foley DP, van den Brand M, Hamburger JN, van der Giessen WJ,
deFeyter PJ, et al. Local intracoronary administration of antisense oligonucleotide against c-myc
for the prevention of in-stent restenosis: results of the randomized investigation by the
Thoraxcenter of antisense DNA using local delivery and IVUS after coronary stenting
(ITALICS) trial. J Am Coll Cardiol 2002; 39: 281-7.
[206]. Nikolova Z, Hof A, Baumlin Y, Hof RP. Efficacy of SDZ RAD compared with
CsA monotherapyand combined Rad/FTY720 treatment in a murine cardiac allotransplantation
model. Transpl Immunol 2001; 9: 43-9.
[207]. Tamai H, Katoh O, Suzuki S, Fujii K, Aizawa T, Takase S, et al. Impact of tranilast
on restenosis after coronary angioplasty: tranilast restenosis following angioplasty trial
(TREAT). Am Heart J 1999; 138: 968-75.
51
Table 1: Attenuation of neointimal thickening by antiproliferative pharmacological
approaches in animal models of vascular proliferative disease.
Agent
References
CVT-313
Animal model
Balloon angioplasty (rat)
Flavopiridol
Balloon angioplasty (rat)
[39]
Rapamycin (Sirolimus)
Balloon angioplasty (rat, pig)
[42-46]
Arterial stent (pig)
Mechanical arterial injury (mouse)
Graft arteriosclerosis (rat)
[48]
[51]
[41,42,44,47]
Everolimus (RAD, certican)
Arterial stent (rabbit)
Graft arteriosclerosis (rat, mouse)
Macrotaline-induced pulmonary
vascular neointimal thickening (rat)
[52]
[54,206]
[53]
Tranilast
Balloon angioplasty (rat)
Graft arteriosclerosis (mouse)
Mechanical arterial injury (mouse)
[64,66]
[67,68]
[69]
Paclitaxel (Taxol)
Balloon angioplasty and arterial stents (rat,
rabbit, pig)
Arterial stent (rabbit)
[56,57,59-62]
Docetaxel
Balloon angioplasty (rabbit)
[63]
Troglitazone
Balloon angioplasty (rat)
[72]
[38]
[58]
52
Table 2: Attenuation of neointimal thickening by antiproliferative gene therapy approaches in animal
models of vascular proliferative disease.
Strategy
Antisense (ODN)
Target gene
CDK2
CDC2
Cyclin B1
CDC2/PCNA
CDC2/PCNA
CDK2
c-myb*
c-myc*
c-myc*
Antisense (retrovirus) Cyclin G1
Animal model
Balloon angioplasty (rat)
Balloon angioplasty (rat)
Balloon angioplasty (rat)
Graft arteriosclerosis (rabbit)
Balloon angioplasty (rat)
Graft arteriosclerosis (mouse)
Balloon angioplasty (pig, rat)
Balloon angioplasty (rat, pig, rabbit)
Graft arteriosclerosis (pig)
References
Balloon angioplasty (rat)
[77]
[34,73]
[34,74]
[74]
[79]
[75]
[78]
[100,101]
[98,102-104]
[105]
Ribozyme
PCNA
TGF-1
Stent (pig)
Balloon angioplasty (rat)
[114]
[115]
‘Decoy’ ODN
E2F
E2F
[118,122]
[119-121]
AP-1
Balloon angioplasty (rat)
Graft arteriosclerosis
(rabbit, mouse, monkey)
Balloon angioplasty (rat, rabbit, minipig)
Oveexpression of
growth suppressors
p21Cip1
p21Cip1
p27Kip1
pRb
RB2/p130
p53
GAX
GATA-6
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)
[133-136]
[138]
[128,137]
[154,155]
[156]
[140,143,153]
[163-165]
[160]
Overexpression of
dominant-negative
mutants
RAS
ERK
JNK
Balloon angioplasty (rat)
Balloon angioplasty (rat)
Balloon angioplasty (rat)
[174,175]
[184]
[184]
[125-127]
* These inhibitory effects might be caused by a nonantisense mechanism [106-110].
53
Table 3: Pharmacological and gene therapy clinical trials for vascular proliferative disease based on
cytostatic strategies.
Trial
RAVEL
Design
Randomized
multicenter,
double-blinded,
placebo-controlled
Randomized
SIRIUS
multicenter,
double-blinded,
placebo-controlled
PREVENT I Randomized,
double-blinded,
single center
PREVENT II Randomized
multicenter,
double-blinded,
placebo-controlled
Randomized
TREAT-1
multicenter,
double-blinded,
placebo-controlled
Randomized
TREAT-2
multicenter,
double-blinded,
placebo-controlled
PRESTO
SCORE
ITALICS
Randomized
multicenter,
double-blinded,
placebo-controlled
Randomized,
multicenter
placebo-controlled
Randomized,
placebo-controlled
Strategy
Rapamycineluting stent
Disease
In-stent
restenosis
Outcome
Prevention
coronary
of
neointimal
proliferation and
late lumen loss
RapamycinIn-stent
Reduction in binary
eluting stent
coronary
restenosis, late lumen
restenosis
loss and repeat
revascularization rates
E2F decoy ODN Autologous
70-74% decreases in
ex vivo
vein graft
the level of positive
transfection
failure after
cell cycle regulators
of vein graft
peripheral
expressed by VSMCs
artery bypass
in the vein, and
reduction in
primary graft failure
E2F decoy ODN Autologous vein Larger patency and
ex vivo
graft failure after inhibition neointimal
transfection
coronary artery
thickening
of vein graft
bypass
Tranilast (oral) Restenosis
Reduced restenosis
after PTCA
in patients with de
novo coronary lesions
(3-month follow up)
Tranilast (oral) Restenosis
Reduced restenosis
after PTCA
in patients with de
novo and restenotic
coronary lesions
(3-month follow up)
Tranilast (oral) Restenosis
No improvement of
after PTCA
restenosis or its
clinical sequelae
(9-month follow up).
7-hexanoyltaxol- In-stent
Reduced neointimal
eluting stent
coronary
growth and larger
restenosis
lumen in patients
with de novo
coronary lesions
c-myc antisense In-stent
No reduction in
ODN delivery coronary
angiographic
after stent
restenosis
restenosis rate
implantation
Refs
[192-194]
[195]
[196]
[4]
[207]
[197]
[198]
[201]
[205]
RAVEL: Randomized study with the sirolimus-eluting bx velocity balloon-expandable stent
SIRIUS: Sirolimus-coated BX velocity balloon-expandable stent in the treatment of
patients with de novo coronary artery lesions
PREVENT: Project of ex-vivo vein graft engineering via transfection
TREAT: Tranilast restenosis following angioplasty trial
PRESTO: Prevention of restenosis with tranilast and its outcomes
SCORE: Study to compare restenosis rate between QueST and QuaDS-QP2
ITALICS: Investigation by the thoraxcenter of antisense DNA using local delivery and IVUS
after coronary stenting
54