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(Circulation. 1997;96:1899-1905.)
© 1997 American Heart Association, Inc.

Articles

Adenoviral Constructs Encoding Phosphorylation-Competent Full-length and Truncated Forms of the Human Retinoblastoma Protein Inhibit Myocyte Proliferation and Neointima Formation

Roy C. Smith, PhD; Ken N. Wills, PhD; Douglas Antelman, PhD; Harris Perlman, BA; Lonn N. Truong, BA; Kevin Krasinski, BA; ; Kenneth Walsh, PhD

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

Top Abstract Introduction Methods Results Discussion References

 
Background The retinoblastoma (Rb) protein is a key cell-cycle regulator that controls entry into the S phase by modulating the activity of the E2F transcription factor. We analyzed the effects of full-length phosphorylation-competent and a mutant truncated form of human Rb for their effects on vascular smooth muscle cell (VSMC) proliferation and neointima formation.

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-terminal–truncated 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

Top Abstract Introduction Methods Results Discussion References

 
The Rb protein is a key modulator of cell-cycle transit because of its ability to control the G₁/S-phase transition (for a review, see Goodrich and Lee¹ ). This activity appears to be dependent on the ability of Rb to modulate the activity of the E2F family of transcriptional regulators that are required for S-phase progression.² In the presence of proliferative signals, the Rb protein is phosphorylated at multiple sites by cyclin/cdk complexes to generate hyperphosphorylated Rb, which is unable to repress E2F-dependent transcription.^{3 4} Rb kinase activity is regulated by a number of cyclin-dependent kinase inhibitors that act to prevent Rb hyperphosphorylation and thereby maintain cells in the G₁ phase.⁵ The proper regulation of Rb is required for the establishment of the postmitotic state in differentiated tissues, and the lack of Rb function can result in tumorigenesis. In myocytes that terminally differentiate, such as skeletal muscle, Rb is maintained in a hypophosphorylated state as a result of high levels of cyclin kinase inhibitor expression.^{6 7 8} Homozygous deletion of the Rb gene occurs in a number of neoplastic diseases, indicating that the regulation of Rb activity is a key checkpoint preventing uncontrolled proliferation. Regulation of Rb phosphorylation may also be a source of malignant progression, and the loss of cdk inhibitors p16 and/or p15 has been shown to occur in a number of malignancies.^{9 10 11 12 13}

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

Top Abstract Introduction Methods Results Discussion References

 
Cell Culture
Rat VSMCs were prepared according to the method of Mader,¹⁵ and human VSMCs were cultured by explant outgrowth from unused portions of saphenous veins from coronary bypass surgery (St Elizabeth's Medical Center, Boston, Mass) according to the method described for internal mammary arteries by Pickering et al.¹⁶ Cultures were used at or below 10 passages and were grown in DMEM containing 15% FBS (GIBCO BRL). Quiescence was induced by incubation for 72 hours in low-mitogen (0.5% FBS) medium.

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 1–encoding 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 I–digested 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 plasmid^{28 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-Thymidine–Incorporation 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-peroxidase–conjugated 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-Catheter–Induced 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

Top Abstract Introduction Methods Results Discussion References

 
Evaluation of Full-length and Mutant Forms of Human Rb as Inhibitors of E2F-Mediated Transcription
To study the effects of the Rb protein on proliferation, a number of mutant forms of Rb were generated by either deletion or site-directed mutagenesis. Fig 1A describes the structure of Rb and indicates regions that were deleted and/or mutated. The ability of the various mutants to inhibit E2F transcriptional activity was measured in an E2F reporter assay. This assay uses a luciferase reporter construct containing four tandem E2F-DNA regulatory elements positioned to support transcription of the luciferase gene. As can be seen in Fig 1B, cotransfection of an E2F expression plasmid, pE2F, produces a 115-fold increase in relative luciferase activity compared with the E2F-luciferase construct alone, and the full-length 110-kD Rb protein decreased this activity by 50% in the fetal rat VSMC line A10. When the cysteine at position 706 of the Rb110 protein is converted to a phenylalanine (H209), Rb-binding proteins are no longer able to associate with Rb.¹⁹ In the E2F reporter assay, this form of Rb does not produce a statistically significant decrease in luciferase activity, suggesting that E2F-binding activity is essential for repression. In contrast, site-directed mutagenesis from serine to alanine residues at phosphorylation site positions 788, 807, and 811 of the full-length Rb construct (Rb110 788/807/811) reduced relative E2F transcriptional activity by 71%. Although this represents a significant repression of E2F transcriptional activity, the greatest inhibitory effect was observed with an amino-truncated Rb (Rb56) protein. As can be seen in Fig 1B, Rb56 produced a 98% reduction of luciferase activity in the E2F reporter assay. The greater transcriptional repression by Rb56 was statistically significant compared with the repression observed with Rb110, as determined by ANOVA analysis (P<.05). The differential effects of Rb56 and Rb110 on E2F-mediated transcription do not appear to result from differences in protein expression levels from the Rb effector plasmids, as determined by Western blot analysis with an anti-Rb antibody (D.A., PhD, unpublished data, 1996). The specificity of Rb56 inhibition was demonstrated by inclusion of the H209 mutation, which does not significantly inhibit luciferase activity. Conversion of the phosphorylation sites at amino acids 788, 807, and 811 in Rb56 did not further reduce luciferase activity (Fig 1B and other data not shown).

View larger version (27K): [in this window] [in a new window]   Figure 1. Effect of human Rb constructs on E2F-mediated expression of luciferase activity. A, The human Rb protein is shown as a linear representation. The A and B E1A/T-antigen binding domains and C-terminal region required for E2F binding2 are indicated. The locations of amino acid (aa) replacements in the effector constructs are indicated by solid arrows. The translational start site of the truncated Rb56 protein is indicated by an open arrow. B, Subconfluent rat fetal smooth muscle cell (A10) cultures were transfected with DNA/DOTAP complexes. After transfection, cultures were incubated for 48 hours in growth medium before whole-cell extracts were prepared. All transfections contained an alkaline phosphatase–expression plasmid (pSV2-AP), an E2F-luciferase reporter plasmid, [p(E2F)x4-E1bTATA-Luc], and an E2F-expression plasmid (pE2F). The control sample labeled "None" lacks pE2F. The control sample labeled "pE2F" lacks only an Rb effector plasmid and represents the basal level of luciferase expression. Test samples also contained the indicated Rb effector plasmid. The results are normalized for transfection efficiency by reporting the ratio of luciferase to alkaline phosphatase activities. The outcome of a representative experiment is shown. All determinations were done in triplicate, and error bars represent the SE. *Statistically significant difference (P<.05) vs pE2F, as determined by ANOVA analysis.

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.

View larger version (18K): [in this window] [in a new window]   Figure 2. Effect of adenovirus constructs expressing full-length Rb (Rb110) or a truncated form (Rb56) of human Rb on (left) rat (rVSMC) and (right) human primary VSMC (hVSMC) proliferation. Quiescent primary VSMC cultures were treated with adenovirus for 24 hours at the indicated MOI (IU/cell) in low-mitogen medium. After infection, the culture was transferred to growth medium to stimulate cell-cycle progression before addition of 3H-thymidine to measure DNA synthesis. Thymidine incorporation was measured by acid precipitation. Results are presented as percent inhibition with respect to a mock-infected control. The results of a representative experiment are shown. Error bars indicate the SE.

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-Gal–infected 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-Gal–infected 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.

View this table: [in this window] [in a new window]   Table 1. Cell-Cycle Analysis of Adenovirus-Infected Cultures of Rat VSMCs

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-Rb56–infected cultures, both the hyperphosphorylated and hypophosphorylated forms of endogenous Rb decreased as Rb56 expression increased. In Ad-Rb110–infected 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.

View larger version (36K): [in this window] [in a new window]   Figure 3. Immunoblot analysis of protein expression in adenovirus-infected cultures of human VSMCs. Quiescent human VSMCs were infected with adenoviral constructs for 24 hours at the indicated MOIs (IU/cell). After infection, cultures were transferred to growth medium for 24 hours before whole-cell extracts were prepared. Ten micrograms of each extract was analyzed by denaturing electrophoresis and transferred to polyvinylidene fluoride membrane. The blot was probed with anti-Rb mouse monoclonal antibody (3C8) and an anti-mouse/horseradish peroxidase secondary antibody. Immune complexes were visualized by chemiluminescence. The positions of the Rb110, Rb56, and phosphorylated form of Rb (Phospho-Rb) proteins are indicated. Analyses of extracts from mitogen-stimulated (P) and quiescent (Q) mock-infected cultures are also shown.

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-Gal–treated or contralateral control arteries. Also shown in Fig 4 are representative hematoxylin-eosin–stained 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-Gal–treated 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

View larger version (49K): [in this window] [in a new window]   Figure 4. Adenoviral delivery of phosphorylation-competent human Rb inhibits neointimal formation. Immunohistochemical analysis of Rb expression was examined in Ad-Rb110–infected arteries (left). Tissue was fixed in formalin and embedded in paraffin, and cross sections were prepared. After deparaffinization and rehydration, sections were incubated with a human-specific anti-Rb mouse monoclonal antibody followed by an anti-mouse/horseradish peroxidase secondary antibody. Immune complexes were visualized with diaminobenzidine (brown) and counterstained with hematoxylin. Shown is a cross section of an artery harvested 48 hours after infection with 1x109 IU of Ad-Rb110. Also shown are representative hematoxylin-eosin–stained carotid artery cross sections obtained from rats 14 days after treatment with 1x109 IU of either Ad-Rb110 or Ad-ß-Gal. Arrows indicate the position of the internal elastic lamina.

View larger version (16K): [in this window] [in a new window]   Figure 5. Effect of phosphorylation-competent human Rb adenoviral constructs on balloon-injury–induced VSMC proliferation in the rat carotid artery model of restenosis. The common carotid arteries of Sprague-Dawley rats were deendothelialized by balloon-catheter abrasion to stimulate neointima formation. Immediately after abrasion, the isolated artery was treated with the indicated adenoviral construct (1x109 IU) for 20 minutes. On completion of the infection period, the virus was removed and blood flow was restored to the treated area. Rats were killed 14 days after treatment, and the treated artery was harvested along with the contralateral untreated vessel, which served as a control. The intimal and medial areas of cross sections from the carotid arteries were determined by quantitative morphometric analysis. *Statistically significant difference (P<.05) vs Ad-ß-Gal–treated control, as determined by an unpaired two-tailed Student's t test.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
By virtue of its central importance in controlling S-phase progression, deletion or inactivation of both Rb alleles appears to be involved in malignant progression in a number of tumors. Consistent with its inactivation during tumorigenesis, Rb reintroduction into both malignant and primary cell lines has been shown to inhibit proliferation.^{37 38 39 40 41} In light of this broad spectrum of activity, Rb is a strong candidate for use as an antiproliferative agent in a number of disorders that are characterized by inappropriate proliferative responses. The proliferation of VSMCs in response to balloon angioplasty may be a relevant example of such an excessive response, and agents that attenuate the proliferation of VSMCs could potentially provide a prophylactic therapy for prevention of restenosis.

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-terminal–truncated 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-terminal–deleted (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

 

CMV = cytomegalovirus FBS = fetal bovine serum IU = infectious units MOI = multiplicity of infection PCR = polymerase chain reaction Rb = retinoblastoma VSMC = vascular smooth muscle cell

Received December 14, 1996; revision received May 12, 1997; accepted May 16, 1997.

	References

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