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(Circulation. 1997;96:628-635.)
© 1997 American Heart Association, Inc.
Articles |
Downregulation of Cyclin G1 Expression by Retrovirus-Mediated Antisense Gene Transfer Inhibits Vascular Smooth Muscle Cell Proliferation and Neointima Formation
From the USC Gene Therapy Laboratories (N.L.Z., P.X.L., E.M.G., W.F.A.), the Divisions of Hematology-Oncology (E.M.G.) and Cardiothoracic Surgery (L.W., V.A.S., F.L.H.), and the Departments of Pediatrics (E.M.G., W.F.A.), Biochemistry (W.F.A.), and Surgery (L.W., V.A.S., F.L.H.), Childrens Hospital of Los Angeles, and the University of Southern California School of Medicine, Los Angeles, Calif.
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Abstract |
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Background The contemporary treatment of coronary athero-occlusive disease by percutaneous transluminal coronary angioplasty is hampered by maladaptive wound healing, resulting in significant failure rates. Morbid sequelae include smooth muscle cell (SMC) hyperplasia and restenosis due to vascular neointima formation.
Methods and Results In this study, we examined the inhibitory effects of a concentrated retroviral vector bearing an antisense cyclin G1 gene on aortic SMC proliferation in vitro and on neointima formation in vivo in a rat carotid injury model of restenosis. Retroviral vectors bearing an antisense cyclin G1 construct inhibited the proliferation of transduced aortic SMCs in 2- to 6-day cultures, concomitant with downregulation of cyclin G1 protein expression and decreased [3H]thymidine incorporation into DNA. Morphological examination showed evidence of cytolysis, giant syncytia formation, and apoptotic changes evidenced by overt cell shrinkage, nuclear fragmentation, and specific immunostaining of nascent 3'-OH DNA ends generated by endonuclease-mediated DNA fragmentation. Pronounced "bystander effects" including neighboring cells were noted in aortic SMCs transduced with the antisense cyclin G1 vector, as determined by quantitative assays and fluorescent labeling of nontransduced cells. In an in vitro tissue injury model, the proliferation and migration of antisense cyclin G1 vector–transduced aortic SMCs were inhibited. Moreover, in vivo delivery of high-titer antisense cyclin G1 vector supernatant to the balloon-injured rat carotid artery in vivo resulted in a significant reduction in neointima formation.
Conclusions These findings represent the first demonstration of the inhibitory effects of an antisense cyclin G1 retroviral vector on nonneoplastic cell proliferation. Taken together, these data affirm the potential utility of antisense cyclin G1 constructs in the development of novel gene therapy approaches to vascular restenosis.
Key Words: angioplasty • muscle, smooth • cyclin G1 • genes
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Introduction |
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The introduction of PTCA represented a major advance in the treatment of vascular stenosis; however, its long-term efficacy is limited by the development of restenosis due to neointima formation in 30% to 50% of patients.1 2 3 Currently, there is no effective treatment for prevention of restenosis in humans. Because the process of luminal narrowing after PTCA of atherosclerotic lesions results, at least in part, from intimal hyperplasia, the control of neointima formation is one of the major goals of contemporary research in vascular biology.4
Vascular myogenic responses and vaso-occlusive sequelae observed after PTCA involve multiple mechanisms.5 6 Denudation and/or damage of the endothelial surface leads to thrombosis, vasoconstriction, and inflammation, which promotes SMC activation and growth. Mathematical analysis of human restenosis underscores the potential importance of cell proliferation.7 Identifiable by histological criteria as early as 5 days after balloon angioplasty,8 the overall incidence of cell proliferation in patients undergoing PTCA is 60% to 80%.9 10 Analysis of proliferating cell nuclear antigen by in situ hybridization found replication rates of 3.8% to 20.7%, and immunocytochemical analysis of restenotic lesions identified the cells in the fibroproliferative tissues as SMCs.11 Detailed kinetic analysis of neointima formation in animal models of balloon injury defined distinct waves of SMC proliferation: (1) a rapid medial SMC proliferation, (2) migration of SMCs across the internal elastic lamina, and (3) replication of SMCs within the intima.12
As important regulators of vascular tone, arterial SMCs are normally maintained in a nonproliferative state within the tunica media. Upon arterial injury, SMCs migrate into the intimal layer of the arterial wall, where they proliferate and produce extracellular matrix components, including collagen, elastin, and proteoglycans. In vascular lesions, SMCs are the predominant cell type, and their migration, accumulation, and proliferation are critical in determining the extent and character of advanced lesions.13 Neointimal SMCs display distinctive phenotypes (vis-à-vis medial SMCs), exhibiting morphological and biochemical properties of embryonic cells.14
Enhanced expression of a number of growth factors has been described in vascular SMCs, atherosclerotic tissue, and/or vascular restenotic lesions, including platelet-derived growth factors,15 transforming growth factor-ßs,16 17 fibroblast growth factors,18 and insulin-like growth factors.19 The high degree of complexity and redundancy in growth factor signaling pathways has encouraged the examination of convergent nuclear events6 or conserved cell cycle regulatory pathways20 21 22 for effective cytostatic therapies. In this study, we report inhibition of aortic SMC proliferation in vitro and neointima formation in vivo in response to a retroviral vector expressing an antisense cyclin G1 construct and provide evidence to support potential mechanisms that contribute to this phenomenon.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Retroviral Vectors, Vector Supernatants, and Producer Cell Lines
The cDNA sequence encoding human cyclin G1 (accession No. X77794) is as originally described by Wu et al.23 The experimental vector bearing the antisense cyclin G1 cDNA24 was packaged in PA317 cells and grown to high-titer clones (vector titer, 1x106 cfu/mL each). The ß-galactosidase and p53 expression vectors were kindly provided as high-titer PA317 packaging cell clones (titers, 5x105 and 2x106 cfu/mL for ß-galactosidase and p53 vectors, respectively) by Genetic Therapy, Inc. The vectors are referred to as G1nBgSvNa, G1p53SvNa.7, and G1aG1SvNa to indicate the order of promoters and coding regions contained in each vector (G1, Moloney murine leukemia virus long terminal repeat sequences; Bg, ß-galactosidase gene; p53, p53 tumor suppressor gene; aG1, antisense human cyclin G1; Sv, SV40 early region enhancer/promoter; and Na, neor gene). The retroviral vector supernatants were further concentrated to a titer of 1x108 cfu/mL by low-speed centrifugation. The vector backbone, G1XSvNa, containing only the SV40 promoter-driven neor gene was used as a control for the effects of transduction and G418 selection.
Cells, Cell Culture Conditions, and Transduction With
Retroviral Vectors
Rat aortic smooth muscle (A10) cells were obtained from American
Type Culture Collection (catalog No. CRL1476) and maintained as
monolayers at a plating density of 2.5x104 cells per well
in DMEM supplemented with 10% FBS (D10). After overnight attachment,
the cells were exposed to 1 mL of the respective retroviral vector in
the presence of Polybrene (8 µg/mL) for 2 hours with periodic
rocking, after which 1 mL of fresh D10 was added to each well.
Forty-eight hours after transduction with the ß-galactosidase vector,
gene transfer efficiency was measured by determining the percentage of
ß-galactosidase–positive cells upon exposure to X-gal
(ß-galactosidase) staining as described previously25 and
visualization by light microscopy.
Analysis of Cell Proliferation, DNA Synthesis, Cyclin G1
Protein Expression, and Apoptosis
To assess the cytostatic effects of retroviral vectors bearing
cell cycle modulators, the SMCs that were transduced with control
vectors or vectors expressing antisense cyclin G1 (or p53) gene(s) were
evaluated for their proliferative potential by counting the number of
viable cells in each culture at serial intervals after transduction.
Values shown represent the mean±SD of triplicate counts. The
effect of cell cycle modulators on DNA synthesis was monitored by the
incorporation of [3H]thymidine into DNA as previously
described.26 Briefly, 24 hours after transduction with the
antisense cyclin G1 or control retroviral vector, the cell cultures
were exposed to [3H]thymidine (1 µCi per well; specific
activity, 6.7 Ci/mmol [1 Ci=37 GBq]; New England Nuclear) for 2
hours. The cells were placed on ice, rinsed twice with cold PBS, and
then rinsed three times with ice-cold 5% TCA. The final TCA rinse was
removed, and the TCA-precipitated material was solubilized with 0.2 mL
of 1 mol/L sodium hydroxide followed by neutralization with an equal
volume of 1 mol/L acetic acid. [3H]Thymidine
incorporation into cellular macromolecules was measured by liquid
scintillation counting and expressed as radioactivity units in
dpm/well. The significance of differences between untreated and
vector-treated groups was determined by ANOVA.
Western analysis of cyclin expression was performed as described previously27 28 with a polyclonal antipeptide antibody recognizing the C-terminal 18 amino acids of human cyclin G1.23 The occurrence of apoptosis in transduced cell cultures was evaluated with the Apoptag Plus in situ detection kit (Oncor), which detects nascent 3'-OH DNA ends generated by endonuclease-mediated DNA fragmentation by enzymatic (terminal deoxynucleotidyl transferase, TdT) addition of digoxigenin-labeled nucleotides followed by immunocytochemical detection of the modified DNA fragments.24
Retrovirus-Mediated Transfer of the Antisense Cyclin G1
Gene in a Rat Carotid Injury Model of Vascular Restenosis
Under general anesthesia (ketamine 10 mg/kg,
rompun 5 mg/kg), in accordance with a protocol approved by the USC
Institutional Animal Care and Use Committee, a 2F Intimax
arterial embolectomy catheter (Applied Medical Resources
Corp) was used to denude the carotid artery endothelium
of Wistar rats (each weighing 400 to 500 g). The catheter was
inserted into the external carotid artery, which was ligated distally,
and passed into the common carotid artery. The balloon was inflated to
a volume of 10 µL and passed three times along the length of the
common carotid artery. After balloon injury, the embolectomy catheter
was removed, and the internal carotid artery was transiently ligated
just distal to the bifurcation. The distal half of the injured segment
was likewise transiently ligated and then exposed to the retroviral
vectors for 15 minutes. Each group of animals received an infusion of
100 µL of concentrated high-titer antisense cyclin G1 vector (n=7) or
a control vector bearing only the neor gene
(n=4), after which the rats were allowed to recover and were fed a
regular mouse/rat diet and water ad libitum. For purposes of analgesia,
the animals were given buprenex 0.2 mg/kg SC every 12 hours for 72
hours after operation. The rats were euthanized 2 weeks after induction
of vascular injury by an overdose of sodium pentobarbital (120 mg/kg
IM), and formalin-fixed sections of both injured and noninjured
contralateral carotid arteries were stained with hematoxylin-eosin and
Sirius red–Verhoeff's elastin stain. Histological
sections were examined by light microscopy, and morphometric evaluation
of the neointima versus media surface areas was made with a
digitizing system; the extent of intimal hyperplasia after vascular
injury is expressed as I:M ratios. The significance of differences
between the I:M ratios of nontreated and vector-treated vessels was
determined by paired t test.29
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Transduction of Aortic SMCs With Retroviral Vectors Bearing Cell Cycle Control Genes
With a nuclear-targeted ß-galactosidase vector (G1nBgSvNa), the apparent transduction efficiency of rat (A10) aortic SMCs was
45%
(see Fig 1A
), which was similar to murine NIH3T3 cells
and somewhat greater than normal human fibroblasts or scar-derived
(keloid) fibroblasts, in which transduction efficiencies of 20% and
30%, respectively, were observed. Transduction of aortic SMCs with
vectors bearing antisense cyclin G1 showed a marked decrease in the
number of viable cells observed at 24 to 144 hours after transduction
compared with transduced cultures containing the empty (control) vector
(Fig 1B). Western analysis confirmed downregulation of cyclin
G1 protein expression in aortic SMCs transduced with antisense cyclin
G1 compared with the control vector24 (Fig 1C).
Proliferation of A10 cells was also inhibited by retrovirus-mediated
overexpression of the p53 tumor suppressor gene in the sense
orientation. Both antisense cyclin G1 and p53 vectors inhibited cell
cycle progression, as determined by the incorporation of
[3H]thymidine (P<.001 for both antisense
cyclin G1 and p53; Fig 1D).
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Antisense Cyclin G1 Induces Degeneration, Multicellular Syncytia
Formation, and Apoptosis in Aortic SMCs
The photomicrographs shown in Fig 2 display the
morphological appearance of aortic SMCs observed by light microscopy at
t=24 hours after transduction with control and antisense cyclin G1
retroviral vectors. As shown in Fig 2A, the cells transduced with the
control vector showed no significant morphological changes. In
contrast, a significant decrease in cell density was observed in
cultures transduced with vectors bearing antisense cyclin G1,
associated with overt degenerative changes, increased multinuclear
syncytium formation, and cytolysis (Fig 2B, 2C, and 2D). Remarkably,
the proportion of cells involved in the syncytia far exceeded the
transduction efficiency as determined by the transduction and
expression of ß-galactosidase. Syncytium formation occurred in A10
cultures transduced with the antisense cyclin G1 vector
supernatants derived from three different high-titer clones,
as well as the p53 vector to some extent, but not in the control
(G1XSvNa) or ß-galactosidase vectors. To further investigate the
mechanisms of cell death, we used a molecular and immunocytochemical
approach to detect the endonuclease-mediated DNA cleavage fragments
that are characteristic of apoptosis. As shown in Fig 2E and 2F, we observed no evidence of apoptosis in cells transduced
with the control vector (Fig 2E); however, a number of
apoptotic cells were observed in the antisense cyclin G1
vector–transduced cultures (Fig 2F). These results indicate that the
cytocidal effects of the antisense cy-clin G1 vector in A10 aortic
SMCs result in part from apoptosis, cell
degeneration, and aberrant syncytium formation.
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Evidence for a Cytocidal "Bystander Effect" in Aortic SMC
Cultures Transduced With Antisense Cyclin G1 Retroviral
Vectors
To confirm that nontransduced cells were indeed incorporated into
the multicellular syncytia found in antisense cyclin G1–transduced
cultures, we loaded nontransduced A10 cells with a fluorescent
marker and overlaid the marked cells on previously transduced cultures
2 hours after washout of the vector supernatant. The incorporation of
nontransduced, fluorescently labeled A10 SMCs into multinuclear
syncytia was clearly evident when these marked cells were overlaid onto
previously transduced A10 cultures (Fig 3A and 3B, low
magnification; 3C and 3D, high magnification; 3A and 3C, phase
contrast; 3B and 3D, UV light). A representative
multinuclear syncytium incorporating cells containing the
fluorescent label is identified by the arrow. Twenty-four hours
after coculture with nontransduced, fluorescently labeled
aortic SMCs, a considerable number of the multinucleated syncytia were
also labeled with the fluorescent dye, indicating that cell
fusion between the transduced and nontransduced cells had occurred.
This finding provides additional evidence of a novel cytocidal
"bystander effect" distinguishable from the classic "bystander
effect" induced by the HStk/GCV system and mediated by gap junctions
present in susceptible cells.30
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The phenomenology of cell fusion was followed over time (Fig 3E
, revealing a significant increase in the number of syncytia that
increased over 4 to 8 hours in aortic SMCs that were transduced with
the antisense cyclin G1 vector (G1aG1SvNa) compared with the cells
transduced with the control vector (G1XSvNa; P<.001). An
appreciable degree of syncytium formation was also noted in cells that
were transduced with the wild-type p53 vector (G1p53SvNa), which also
produced marked cytostasis in A10 cells. However, the number of
syncytia observed in p53-transduced cells was significantly less than
that observed in antisense cyclin G1–transduced cells at 8, 12, and 24
hours (P<.001).
Antisense Cyclin G1 Vector Inhibits Proliferation and Migration of
Aortic SMCs in an In Vitro "Tissue" Injury Model
High-density (confluent) monolayer cultures of A10 SMCs exhibiting
contact inhibition of cell growth can be stimulated to proliferate
along a track of cell/tissue disturbance exhibiting a
characteristic "wound-healing" response over a period of 7 days.
Fig 4A shows high-density cultures of aortic SMCs
scraped with a 200-µL pipette tip to create a 1-mm track devoid of
cells. Fig 4B shows the appearance of the "wound" margin
immediately upon scraping and washing to remove the detached cells. As
shown in Fig 4C, subsequent transduction of the cell cultures (at t=24
hours) with a nuclear-targeted ß-galactosidase vector was greatest at
the margins of the "wound," an area of activated SMC
proliferation. Fig 4D shows proliferation and migration of aortic SMCs
into the wound track at t=24 hours after injury. In contrast,
apoptotic and other degenerative changes were observed in the
SMCs that were transduced with the antisense cyclin G1 vector (Fig 4E).
Notably, these degenerative changes were marked by multicellular
syncytia formation that was not observed in either the control or
ß-galactosidase vector. Furthermore, cell proliferation and overt
cell migration into the wound track was markedly reduced in the
antisense cyclin G1–transduced cell cultures, evidenced by delayed
closure of the wound track (7 days) compared with the control
vector–treated cultures (3 days).
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Inhibition of Neointima Formation In Vivo by Infusion
of High-Titer Antisense Cyclin G1 Vector Supernatant
Previous studies demonstrated direct transfer of recombinant
marker genes into the arterial wall by retroviral vectors
with viral titers of 104 to 106
particles/mL,31 and a number of studies have demonstrated
the efficacy of cytostatic gene therapies delivered by other methods in
animal models of vascular restenosis (see "Discussion").
In this study, we generated high-titer retroviral vector supernatants
(viral titer, 1x108 cfu/mL) to test the efficacy of
antisense cyclin G1 delivered by highly concentrated retroviral vectors
in the rat carotid injury model of restenosis.
Histological examination of stained sections obtained
from balloon-injured untreated arteries showed substantial
neointima formation at t=2 weeks, as evidenced by invasion
of the tunica intima by proliferating vascular SMCs (Fig 5A and 5C). In contrast, injured arterial segments
that were treated with high-titer antisense cyclin G1 vector
supernatants showed a significant reduction in neointima
formation (Fig 5B and 5D). Morphometric analysis confirmed
significant inhibition in neointima formation in injured
carotid arteries that were treated with the antisense cyclin G1
retroviral vector (I:M ratio, 0.4±0.4 SD) compared with the untreated
arterial segments (I:M ratio, 1.1±0.4; P<.001;
Fig 5G). In control studies, there was no difference between the extent
of neointima formation in nontreated arterial
segments (I:M ratio, 1.3±0.5) compared with high-titer vectors
containing only the neor gene (I:M ratio,
1.5±0.2).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Clinical trials based on the molecular blockade of identified growth factors and/or growth factor receptors implicated in the pathogenesis of intimal hyperplasia have not proved to be effective vehicles for cytostatic vascular therapy.32 Thus, it has been suggested that approaches that target intracellular signaling cascades shared by many growth-regulatory molecules may be more strategic.33 Accordingly, novel gene therapy approaches to inhibit SMC proliferation and neointima formation have recently focused on cell cycle control mechanisms. Indeed, antisense approaches against cell cycle regulatory genes have been shown to be remarkably effective in limiting neointimal hyperplasia in animal models of lesion formation after both bypass surgery34 and balloon angioplasty. A single intraluminal delivery of antisense Cdc2 kinase or Cdk2 kinase produced significant inhibition of neointimal hyperplasia.20 21 35 An adenoviral vector bearing a nonphosphorylatable, constitutively active form of retinoblastoma protein was also reported to inhibit SMC proliferation and neointima formation after balloon angioplasty.36 Molecular strategies directed against E2F have also been developed, because the concerted induction of numerous cell cycle regulatory genes is regulated by this transcription factor. Oligonucleotides containing the E2F cis element sequence function as "decoys" that bind E2F within the cell and inhibit neointimal lesion formation in vivo.37 Further support for the concept of cytostatic gene therapy based on the inhibition of cell cycle control enzymes is provided by recent findings that rapamycin, which inhibits the activation of cell division/cycle enzymes,38 39 40 also inhibits vascular lesion formation in both rat and porcine models.41 42
Cyclin G1 is a member of the so-called G1 family of cyclins, which act in concert with cyclin-dependent protein kinases during the G1 phase of the cell cycle.28 43 Induced in early G1 and suspected to participate in the molecular mechanisms of cell activation,23 cyclin G1 appears to be a transcriptional target of the p53 tumor suppressor gene.44 Cyclin G1 overexpression was first linked to cancer23 and, more recently, downregulation of cyclin G1 expression by retroviral vectors bearing antisense CYCG1 was reported to inhibit the growth and survival of human osteosarcoma (MG-63) cells.24 These observations encouraged us to further investigate the potential utility of the antisense cyclin G1 vectors in other clinical conditions in which uncontrolled cell proliferation was a prominent feature.
In the present study, we examined the effects of retroviral vectors bearing an antisense cyclin G1 construct on the proliferation of A10 rat aortic SMCs. Retroviral vectors bearing the antisense cyclin G1 gene, as well as the p53 gene, in sense orientation inhibited the survival and proliferation of transduced A10 cells in 2- to 6-day cultures. Cytostasis was associated with decreased DNA synthesis and downregulation of cyclin G1 in vascular SMCs transduced with the antisense cyclin G1 vector compared with those transduced with the control vector. Morphological examination of the transduced SMCs revealed cytolysis, giant syncytia formation, and overt apoptotic changes evidenced by cell shrinkage, nuclear fragmentation, and chromatin condensation observed in both antisense cyclin G1 vector– and p53 vector–transduced A10 cells. However, the numbers of multinuclear syncytia were found to be significantly higher in the cell cultures treated with the antisense cyclin G1 vector. Pronounced "bystander effects" were noted in A10 cells transduced with the antisense cyclin G1 vector as determined by quantitative cell fusion assays and the fluorescent labeling of nontransduced cells. These findings indicate that the antisense cyclin G1 vector induces a "fusion-promoting factor," possibly a protease or glycosylase, that facilitates cell fusion and syncytia formation, perhaps by augmenting mechanisms related to the fusogenic properties of the MoMuLV envelope protein.45
Cytostatic gene therapies for restenosis show promise of
additional therapeutic consequences in that the inhibition of cell
cycle regulatory genes is reported to trigger vascular cell
apoptosis.46 In mitotically activated
SMCs, as in osteosarcoma cells,24 the cytotoxicity of the
cyclin G1 blockade is attributable, at least in part, to the activation
of an apoptotic pathway (see Fig 2F). Furthermore, the
induction of cell cycle arrest in some circumstances also appears to
inhibit SMC migration and extracellular matrix
production.47 In the in vitro "tissue
injury" model, both the proliferation and migration of A10 cells
that were transduced with the antisense cyclin G1 vector were inhibited
in the area of cell injury (see Fig 4E). Taken together with the
observations of marked cytotoxicity, cell cycle blockade, and
multicellular syncytia formation, these findings lend additional
support for the concept that cyclin G1 may represent a
strategic locus for therapeutic intervention in the management of
proliferative disorders.
Once a potential therapeutic gene has been identified, the challenge remains to deliver the gene transfer vector efficiently to the appropriate physiological site. In the case of balloon angioplasty, both the denudation of the endothelial lining and the mitogenic activation of neighboring SMCs provide favorable conditions for the delivery of retroviral vectors, because the therapeutic genes delivered by retroviral vectors are expressed preferentially in mitotically active cells. In the present study, we generated very-high-titer supernatants (108 cfu/mL) to enhance the transduction efficiency of vascular SMCs and hence the efficacy of retroviral vectors in this experimental model of restenosis. Indeed, the in vitro studies of retroviral vector–mediated gene delivery in embryonic A10 SMCs may be particularly relevant to the physiology of restenosis, because numerous reports have indicated that embryonic and neointimal SMCs exhibit similar responses to mitogenic signals.14 This study in the rat carotid artery injury model of restenosis demonstrates the efficacy of this approach: Sections of balloon-injured carotid arteries that were treated with an infusion of highly concentrated (108 cfu/mL) antisense cyclin G1 retroviral vector supernatant showed a significant reduction in neointima formation. Taken together, these data support the utility of retroviral vectors bearing cyclin G1, alone or in combination with p53 or the now-classic HStk/GCV approach, in the development of novel gene therapy strategies to combat vascular restenosis.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by grant GM-49715 from the National Institutes of Health, Institute of General Medical Sciences, awarded to Dr Hall.
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Footnotes |
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Reprint requests to Frederick L. Hall, PhD, Division of Cardiothoracic Surgery, Childrens Hospital Los Angeles, MS 126, Los Angeles, CA 90027.
Received October 9, 1996; revision received January 15, 1997; accepted January 22, 1997.
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