(Circulation. 1997;96:1899-1905.)
© 1997 American Heart Association, Inc.
Articles |
From the Division of Cardiovascular Research, Department of Medicine, Tufts University School of Medicine, St. Elizabeth's Medical Center (R.C.S., H.P., L.N.T., K.K., K.W.), Boston, Mass; CANJI, Inc (K.N.W., D.A.), San Diego, Calif; and the Program in Cell, Molecular and Developmental Biology, Sackler School of Biomedical Sciences, Tufts University (H.P., K.W.), Boston, Mass.
Correspondence to Kenneth Walsh, Division of Cardiovascular Research, St Elizabeth's Medical Center, 736 Cambridge St, Boston, MA 02135. E-mail kwalsh{at}opal.tufts.edu
| Abstract |
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Methods and Results A number of mutant forms, both phosphorylation competent and incompetent, of human Rb protein were evaluated for their ability to inhibit E2F activity. The results of these assays indicated that a phosphorylation competent, amino-terminaltruncated Rb protein (Rb56) was a particularly potent inhibitor of E2F-mediated transcription relative to the full-length Rb construct (Rb110). Adenoviral constructs containing Rb56 or Rb110 expressed their respective Rb forms in VSMCs, as determined by Western immunoblot analysis, and were similar in their abilities to arrest the cell cycle, as determined by assays of 3H-thymidine incorporation and by flow cytometric analyses. When examined for their effect on neointima formation after balloon injury of the rat carotid artery, both full-length and truncated forms of Rb inhibited formation of this VSMC-derived lesion.
Conclusions These analyses revealed that the maintenance of high levels of phosphorylation-competent human Rb, either full-length or truncated forms, in VSMCs is an effective method of modulating the extent of intimal hyperplasia that occurs after balloon-induced vascular injury.
Key Words: angioplasty genes carotid arteries muscle, smooth stenosis
| Introduction |
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Recently, Chang et al14 showed that a nonphosphorylatable, constitutively active form of murine Rb inhibited VSMC proliferation and reduced neointima formation in the injured rat carotid artery and porcine femoral artery models, suggesting that similar regulatory networks may underlie carcinogenesis and proliferative vessel-wall disorders. In the study of Chang et al,14 the effects of wild-type Rb were not analyzed. In the present study, we evaluate the ability of human Rb, both the phosphorylation-competent full-length form and a truncated form, to inhibit E2F transcriptional activity, VSMC proliferation, and neointima formation in the rat balloon-injury model. The results presented herein suggest that adenovirus-mediated Rb inhibition of VSMC proliferation is, to a large extent, dependent on the quantity of exogenous protein produced.
| Methods |
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Construction of Rb and E2F Expression Plasmids
A partial cDNA (amino acids 95 through 437) of the
E2F gene was obtained from pGEX2T-E2F-XH917 via
PCR amplification. The amplified fragment was digested with
HindIII and subcloned into pCMV to generate pCMV-E2F. The
full-length Rb110 cDNA was obtained from pA9-RB18 by
digestion with Sac I/HindIII and subcloned into
pCMV to generate pCMV-RB110. The RB110-H209 mutant contains the change
TGT (Cys) to GCA (Phe) at position 706 as described
previously.19 The serine-to-alanine mutations in Rb110
788/807/811 were generated by subcloning an EcoRI fragment
of the cDNA into M13mp18 and performing site-directed mutagenesis as
described previously.20 Codon changes used were AGC to GCC
(788), TCA to GCA (807), and AGT to GCT (811). The Rb56 construct was
generated by PCR amplification of the pA9-RB plasmid with the use of a
5 primer (GTCGACATGAACACTATC) containing a Sal I
restriction site and start codon and a 3 primer containing a
HindIII restriction site. The resulting PCR product was
subcloned into pCMV to obtain pCMV-RB56. The H209 and 788/807/811 Rb56
constructs were generated from the corresponding Rb110 constructs.
Construction of Adenovirus Vectors and Their Amplification in
Culture
The recombinant adenoviruses used in the present studies are
based on serotype 5 human adenovirus from which the viral early region
1encoding E1a, E1b, and pIX proteins have been deleted. This
adenovirus is limited to propagation in 293 cells that produce the Ad5
E1 gene products required for replication. Transfer plasmids
encoding either full-length or truncated Rb were generated from
pACN21 and were in turn used to construct the recombinant
adenoviruses. Either a full-length Rb cDNA (amnio acids 1 through 928),
subcloned as a 2.8-kilobase Xba I, BamHI fragment
from the plasmid pETRbc,22 or a truncated version of Rb
(amino acids 381 through 928), subcloned as a 1.7-kilobase
Xba I, BamHI cDNA fragment, was placed downstream
from the CMV promoter/enhancer and the Ad2 tripartite leader cDNA of
the plasmid pACN. These plasmids were subsequently linearized with
EcoRI and cotransfected (CaPO4; Stratagene) with
either the isolated Cla Idigested large fragment of
H5ilE423 to make Ad-Rb56 (ACN56) containing a partial E4
deletion or with the large fragment from a hybrid virus of
dl32724 and H5ilE4 to create Ad-Rb110 (ACNRb), which
contains deletions in both the E3 and E4 regions of the vector. The
control virus, Ad-ß-Gal (ACBGL), contains the bacterial
ß-galactosidase gene downstream from the CMV
promoter/enhancer.25 Isolation and initial purification of
the viruses were performed by standard methods,26 and
purified virus was prepared according to the method of Huyghe et
al.27
Measurement of the Effects of Rb Variants on an E2F Expression
System in Transiently Transfected Cells
A10 cells were plated at 50% confluence on 10-cm dishes in
growth medium. Transfections used a total of 5 µg of DNA with 90 µg
of DOTAP (Boehringer Mannheim) in Opti-MEM. The DNA-lipid mixture was
incubated for 20 minutes at room temperature in serum-free Opti-MEM,
after which cells were transfected for 5 hours at 37°C. After
transfection, growth medium was added and the cells were incubated for
an additional 48 hours before being harvested.
Cells were cotransfected with 2.4 µg of the test plasmid and 2 µg of an E2F-luciferase reporter plasmid, (E2F)x4-E1bTATA-Luc, containing the PvuII/Sac I fragment from the promoter region of the (E2F)x4-E1bTATA-CAT plasmid28 29 cloned into the Sma I/Sac I site of the pGL2-Basic plasmid (Promega) and positioned to drive the expression of the luciferase gene. Included in the transfection mixture was 0.1 µg of a plasmid (pE2F) containing the E2F1 structural gene under control of the CMV promoter/enhancer, which was necessary to provide expression of sufficient E2F protein to drive the assay. The transfection mixture also contained 0.6 µg of pSV2-AP30 containing the bacterial alkaline phosphatase gene under control of the SV40 promoter/enhancer as an internal control for transfection frequency. The Rb expression plasmids have the various Rb gene constructs downstream from the CMV promoter/enhancer.
Alkaline phosphatase activity of transfected cells was measured with the use of CSPD chemiluminescent substrate (Tropix), and luciferase activity was measured with the Luciferase Assay System from Promega. Activity was reported as relative light units based on the ratio of luciferase to alkaline phosphatase activity.
Measurement of the Effects of Adenovirus Infection on Cellular
Proliferation by the 3H-ThymidineIncorporation
Assay
Quiescent cultures were infected in low-mitogen medium (0.5%
FBS) for 24 hours. After infection, the cultures were transferred to
growth medium and incubated for 14 hours (24 hours for human VSMCs)
to allow entry into the S phase. Cell proliferation was determined by
thymidine incorporation in medium containing 3 µCi/mL
[methyl-3H]-thymidine (6.7 Ci/mmol, NET-027; DuPont NEN)
for 4 hours (12 hours for human VSMCs). Incorporated label was
determined as acid-precipitable material (10% trichloroacetic acid) by
liquid scintillation counting in Scintiverse II (Fisher) with a Beckman
LS 5000TD scintillation counter. All determinations were done in
triplicate.
Flow Cytometric Analysis of Cell-Cycle Progression in
Adenovirus-Infected Cultures
Quiescent rat primary VSMCs were infected for 12 hours at 750
MOI (IU/cell) in low-mitogen medium. After infection, the adenovirus
solution was removed, and the cultures were incubated for 12 hours in
low-mitogen medium before being transferred to growth medium (10% FBS)
for 14 hours to stimulate cell-cycle progression. The cultures were
harvested by trypsinization and fixed in 70% ethanol for 30 minutes at
-20°C. The cells were then stained for DNA content (PBS, 0.1%
Triton X-100, 0.1 mmol/L EDTA, 0.05 µg/mL RNase A,
and 50 µg/mL propidium iodide), and the cell-cycle profile was
determined by flow cytometric analysis with the use of a Becton
Dickinson Vantage flow cytometer and Lysis II cell-cycle
analysis software. Flow cytometry was conducted at the Core
Flow Cytometry Facility of the Dana-Farber Cancer Institute, Boston,
Mass.
Immunoblot Analysis of Rb Expression
in Adenovirus-Infected Cultures
Quiescent primary human VSMC cultures were infected with virus
for 24 hours in low-mitogen medium. After infection, the cultures were
transferred to growth medium (15% FBS) for 24 hours. Whole-cell
extracts were prepared from these cultures,31 and protein
concentrations were determined from the optical densities at 280 and
260 nm. Ten micrograms of each extract was analyzed by SDS-PAGE
on a 7.5% polyacrylamide gel under reducing conditions
(ß-mercaptoethanol). Proteins were transferred by the semidry method
to a polyvinylidene fluoride membrane (Immobilon-P, Millipore) and the
membrane was probed with an anti-human Rb mouse monoclonal antibody
(3C8; 1.7 µg/mL)32 in 2% milk/TBS-T (10
mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, and 0.05%
Tween-20). The secondary antibody (horseradish-peroxidaseconjugated
anti-mouse; Amersham) was used at 1:4000 dilution, and complexes were
visualized with Enhanced Chemiluminescence (ECL) reagent (Amersham).
Exposures were converted to a digital image by use of an Eagle Eye II
video imaging system with Eagle Sight software (version 2.0;
Stratagene), and band intensities were determined by use of One-Dscan
software (version 1.0; Scanalytics).
Measurement of Adenovirus Effects on
Balloon-CatheterInduced Injury in the Rat Carotid Artery Model
of Restenosis
This model of balloon injury was based on that described by
Clowes et al.33 34 Briefly, the left common carotid artery
of a male Sprague-Dawley rat weighing 400 to 500 g was subjected
to a distending, deendothelializing injury by abrasion
with an inflated 2F embolectomy catheter inserted via the external
carotid artery. The injured segment of the artery was then incubated
with adenovirus (1x109 IU) diluted to 100 µL with 15%
(wt/vol) Poloxamer 407 (BASF)35 for 20 minutes, after
which the viral infusion was withdrawn and the external carotid artery
was ligated. This experimental protocol was approved by the
Institutional Animal Care and Use Committee and complied with the
Guide for the Care and Use of Laboratory Animals (NIH
publication No. 86-23, revised 1985).
Rats were killed 14 days after treatment. The injured segment of the left common carotid artery was dissected free from the surrounding tissue and fixed in 100% methanol until embedded in paraffin. Several 4-µm sections were cut from each specimen. Sections were stained with either hematoxylin and eosin or Richardson's combination elastic-trichrome stain for conventional light microscopic analysis.
Histological images of cross sections were projected onto a digitizing board (Summagraphics), and the intimal, medial, and luminal areas were measured by quantitative morphometric analysis with a computerized sketching program (Macmeasure version 1.9; National Institute of Mental Health).
Immunohistochemical Analysis of Rb Expression in
Adenovirus-Infected Tissue
Adenovirus-treated carotid arteries were harvested from rats 2
days after balloon injury and infection. Tissue was fixed in
phosphate-buffered formalin and embedded in paraffin. Cross sections
(4 µm) were cut and dewaxed through xylene and graded alcohols.
Endogenous peroxidase was quenched with 1% hydrogen
peroxide for 30 minutes. Antigen retrieval was performed in 10
mmol/L sodium citrate buffer, pH 6.0, at 95°C for 10 minutes.
A monoclonal anti-Rb antibody (AB-5; Oncogene Sciences) was applied (10
µg/mL) at 4°C for 24 hours. Secondary antibody was applied
from the Unitect Mouse Immunohistochemistry Kit (Oncogene Sciences)
according to the manufacturer's instructions. Antibody complexes were
visualized by use of 3,3-diaminobenzidine (DAB; Vector
Laboratories), and slides were counterstained with hematoxylin.
Statistical Analysis
Differences between groups were analyzed by use of
either an unpaired two-tailed Student's t test or
single-factor ANOVA. Statistical significance was assumed when the
probability of a type I error was <.05. Statistical tests were
conducted with the use of Statview software from Abacus Concepts, Inc
(Macintosh version 4.1).
| Results |
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Inhibition of VSMC Proliferation by Adenoviral Constructs of
Full-length or a Truncated Form of Human Rb
To compare the efficacy of Rb110 and Rb56 proteins as
inhibitors of proliferation, we constructed
replication-deficient adenoviral vectors expressing these proteins
under control of the CMV promoter. When these vectors were tested for
the ability to inhibit the mitogen-stimulated proliferation of rat and
human VSMCs (Fig 2
), as measured by
3H-thymidine incorporation, we observed a potent inhibition
with both the full-length Rb110 (Ad-Rb110) and the truncated Rb56
(Ad-Rb56) viruses at MOIs >100. The dose-response curves were similar
for both forms of Rb in human and rat VSMCs. Ad-ß-Gal did not inhibit
rat or human VSMC proliferation to a significant extent, as can also be
seen in Fig 2
.
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Flow Cytometric Analysis of Cell-Cycle Progression in
Transduced Rat Primary VSMCs
The ability of the adenoviral constructs to inhibit rat aorta VSMC
proliferation is reflected by their ability to induce cell-cycle
arrest, as shown in the Table
. The
quiescent mock-infected culture displayed 70.0% of cells in the
G0/G1 phase of the cell cycle.
Proliferating mock-infected and Ad-ß-Galinfected cultures had a
similar number of cells in the G0/G1
phase (43.9% and 45.9%, respectively). However, when the cells were
infected with the adenovirus encoding the full-length Rb (Ad-Rb110),
70.5% of the culture was in the G0/G1
phase, and in cultures infected with amino-truncated Rb (Ad-Rb56),
76.7% of the culture was in G0/G1.
Cultures transduced with either Rb vector displayed correspondingly
lower proportions of cells in the S and G2/M phases
compared with mock- or Ad-ß-Galinfected cells. These data further
demonstrate that both the wild-type Rb110 and the amino-truncated Rb56
are similar in their cell-cycle regulatory properties in VSMCs.
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Western Immunoblot Analysis of Human Rb
Expression by Adenovirus-Infected Cells
Ad-Rb56 and Ad-Rb110 expressed their respective proteins as
indicated by Western immunoblot analysis of
whole-cell extracts from infected human VSMCs, as shown in Fig 3
. Infection at MOIs >100 with either
Ad-Rb110 or Ad-Rb56 resulted in substantial overexpression of the
recombinant Rb proteins relative to endogenous Rb. The
hypophosphorylated and
hyperphosphorylated forms of endogenous Rb
could be resolved, and the slower-migrating
hyperphosphorylated form was clearly present in the
uninfected proliferating culture but was absent in the quiescent
(serum-starved) culture. Infection with the Ad-ß-Gal control virus
appeared to decrease the extent of endogenous Rb
phosphorylation at an MOI >100. This can be correlated
with a slight inhibition of human VSMC proliferation at these MOIs (Fig 2
). In Ad-Rb56infected cultures, both the
hyperphosphorylated and
hypophosphorylated forms of endogenous Rb
decreased as Rb56 expression increased. In Ad-Rb110infected
cultures, we also observed a marked decrease in
hyperphosphorylated Rb expression with increasing
levels of virally encoded Rb expression. These data demonstrate that
the replication-deficient adenoviral constructs can substantially
overexpress the full-length and truncated forms of Rb relative to the
expression of endogenous Rb in VSMCs. Furthermore, these
data reveal that the level of Rb phosphorylation is
sensitive to overall levels of recombinant Rb expression, either
full-length or truncated, and can also be affected to a lesser extent
by transduction with control virus.
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Effect of Human Rb Adenoviral Constructs on Neointima
Formation in the Rat Carotid Artery Model of Restenosis
The ability of the Rb adenoviral constructs to inhibit VSMC
proliferation in vitro led us to test their ability to inhibit the
hyperproliferation of VSMCs in the rat carotid artery model of
stenosis. Chang et al14 reported that an
adenovirus construct encoding a
phosphorylation-resistant form of murine Rb was
able to inhibit neointima formation relative to an
Ad-ß-Gal construct in this model. In Figs 4
and 5
,
the results with both phosphorylation-competent human
Rb adenoviral constructs (Ad-Rb110 and Ad-Rb56) and the Ad-ß-Gal
construct are shown. To observe the extent of adenoviral transduction,
we treated injured arteries with either Ad-Rb110 or Ad-ß-Gal and
killed the animals 2 days after infection to harvest both the treated
carotid artery and the contralateral untreated artery as a control.
Tissues sections were stained for the presence of human Rb with a
human-specific monoclonal anti-Rb antibody, and, as shown in Fig 4
, extensive staining of the most luminal layers of smooth muscle cells
was observed. Positive staining for human Rb was not observed in either
the Ad-ß-Galtreated or contralateral control arteries. Also shown
in Fig 4
are representative hematoxylin-eosinstained
cross sections from adenovirus-treated arteries 14 days after
treatment. As can be observed, Ad-Rb-110 inhibited
neointima formation in this system compared with the lesion
obtained in an Ad-ß-Galtreated artery. For the full-length Ad-Rb110
construct, a 39% reduction (P=.015) in the intima/media
ratio was obtained compared with Ad-ß-Gal (Fig 5
). Similarly, a 43%
reduction (P=.0025) was observed with the Ad-Rb56 construct.
Neointima formation in vessels infected with the Ad-ß-Gal
construct were equivalent to the values observed for saline treatment
of injured vessels.36
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| Discussion |
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The goal of our study was to analyze the regulatory properties of various forms of human Rb in VSMCs. We initiated this study by examining the effects of mutations in Rb that were anticipated to modulate the E2F binding activity. Full-length phosphorylation-competent Rb (Rb110), full-length Rb with mutations at a subset of phosphorylation sites (Rb110 788/807/811), and an amino-terminaltruncated form of Rb (Rb56) all inhibited E2F-mediated transcription from a luciferase reporter construct. The Rb56 construct appeared most potent in inhibiting E2F-mediated transcription. Rb forms with an inactivating mutation (H209) had no effect on transcription. On the basis of these results, we created adenoviral constructs that expressed Rb protein in either the full-length (Ad-Rb110) or the amino-terminaldeleted (Ad-Rb56) forms and compared their ability to inhibit VSMC proliferation.
When examined for their effect on VSMC proliferation in vitro, the full-length Rb110 and truncated Rb56 forms were surprisingly similar in their growth-inhibitory activities. Both the Rb110 and the Rb56 adenoviral constructs were able to bring about similar levels of G0/G1 arrest in rat VSMCs compared with the Ad-ß-Gal or mock-infected controls, consistent with the notion that Rb functions as an important regulator of the G1- to S-phase transition in VSMCs. The ability of full-length Ad-Rb110 to inhibit VSMC proliferation indicates that high levels of phosphorylation-competent Rb are sufficient to inhibit cell-cycle progression. The Rb adenoviral constructs were also compared in their abilities to inhibit neointima formation in the rat carotid artery model of stenosis. Both constructs were able to significantly reduce the intima/media ratio. Two weeks after injury, Ad-Rb56 reduced the intima/media ratio by 43%, whereas Ad-Rb110 had a similar reduction of 39%. Our results with human Rb constructs compare favorably with the reduction in intima/media ratio obtained by Chang et al14 (42%) using an adenovirus construct that encodes a constitutively active, nonphosphorylatable form of murine Rb. Thus, whereas others have sought to control the level of Rb activity by delivering a form of Rb protein that is insensitive to hyperphosphorylation, the results presented herein suggest that overexpression of phosphorylation-competent Rb is sufficient for inhibition of neointima formation.
Together, these observations indicate that the full-length phosphorylation-competent human form of Rb can function in a similar manner as a mutant nonphosphorylatable murine Rb in the rat carotid artery model system of restenosis. The use of human Rb in clinical applications should lower the possibility of an immune response that may be anticipated when cross-species gene transfer is conducted. For this reason, we believe that wild-type human Rb represents a conservative and practical choice for preventing the VSMC hyperproliferation that occurs after balloon angioplasty.
| Selected Abbreviations and Acronyms |
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Received December 14, 1996; revision received May 12, 1997; accepted May 16, 1997.
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