Adenoviral RB2/p130 Gene Transfer Inhibits Smooth Muscle Cell Proliferation and Prevents Restenosis After Angioplasty
Abstract—Smooth muscle cell (SMC) proliferation that results in neointima formation is implicated in the pathogenesis of atherosclerotic plaques and accounts for the high rates of restenosis that occur after percutaneous transluminal coronary angioplasty, a widespread treatment for coronary artery disease. Endothelial lesions trigger intense proliferative signals to the SMCs of the subintima, stimulating their reentry into the cell cycle from a resting G0 state, resulting in neointima formation and vascular occlusion. Cellular proliferation is negatively controlled by growth-regulatory or tumor-suppressor genes, or both, such as the retinoblastoma gene family members (RB/p105, p107, RB2/p130). In the present study, we show that RB2/p130 inhibited SMC proliferation in vitro and in vivo. We used the rat carotid artery model of restenosis to demonstrate that adenovirus-mediated localized arterial transduction of RB2/p130 at the time of angioplasty significantly reduced neointimal hyperplasia and prevented restenosis. Furthermore, the ability of pRb2/p130 to block proliferation correlated with its ability to bind and sequester the E2F family of transcription factors, which are important mediators of cell cycle progression. These results imply that RB2/p130 could be an important target for vascular gene therapy.
It is well documented that chronic or acute injury to the arterial wall, such as that occurring with percutaneous transluminal coronary angioplasty (PTCA), induces the expression of a variety of growth factors and inflammatory cytokines that stimulate smooth muscle cell (SMC) proliferation and migration from the media into the intima, resulting in neointima formation and eventual restenosis.1 Inhibition of neointima formation should greatly improve the effectiveness of PTCA in the long-term management of coronary artery disease. Numerous growth factors induce SMC proliferation through a variety of signal transduction pathways in vitro and in vivo.2 This evidence suggests that critical regulatory proteins of the cell cycle machinery should be targeted instead of the upstream signal transduction molecules for effective cytostatic therapy of vascular proliferative disorders.3 4 5
Various strategies have been used in animal models to prevent restenosis, including the transfer of the herpes simplex virus thymidine kinase associated with ganciclovir and the transduction of cell cycle regulatory genes such as RB/p105.4 5
In particular, the retinoblastoma family proteins (pRb/p105, p107, and pRb2/p130) are excellent candidates for vascular disease gene therapy. They are nuclear phosphoproteins with growth-suppressive properties that interact with specific members of the E2F transcription factor family (E2F-1 to E2F-5) and are regulated by phosphorylation/dephosphorylation events in a cell cycle–dependent manner.6 7 Previous studies show that the induction of pRb2/p130 expression growth arrests proliferating cells in the G0/G1 phase of the cell cycle through direct interaction with and regulation of the activity of the cell cycle machinery.8 9 10 Furthermore, the induction of pRb2/p130 expression inhibits cellular proliferation in certain cell lines that are refractory to the effects of Rb family members pRb/p105 and p107.8 9 In this study, we demonstrate that adenovirus-mediated transduction of RB2/p130 blocks SMC proliferation in vivo and in vitro. Moreover, we show that RB2/p130 preferentially interacts with and sequesters the growth-promoting transcriptional activity of E2F-4 in SMCs.
Adenoviruses have been shown to serve as effective and efficient vectors to deliver transgenes to cells in vivo. We decided to use adenovirus-mediated RB2/p130 gene transfer to hold in check the proliferative capacity of the arterial smooth muscle subsequent to acute injury to prevent restenosis after angioplasty.
The growth-inhibitory effects of activators of the cAMP-dependent protein kinase A or of dominant negative ras proteins have been demonstrated in SMCs.11 12 With a similar approach, we determined the effects of adenovirus-mediated transduction of RB2/p130 on the proliferative capacity of SMCs both in vitro and in vivo.
Materials and Methods
Primary human embryonic kidney 293 cells (American Type Culture Collection) and pulmonary artery smooth muscle (PASM) cells have been previously described.14
Adenoviral Production and Transduction
Ad-CMV-RB2/p130, Ad-CMV, and Ad-CMV-β-Gal were generated and purified with CsCl as previously described.13 15 16 A viral titer of 22×109 plague-forming units (pfu)/mL was determined with a plaque assay for the Ad-CMV and Ad-CMV-Rb2/p130 viruses. A viral titer of 40×109 pfu/mL was determined for the Ad-β-Gal virus.
Determination of Growth Rates and Western Blotting
PASM cells were plated at a density of 1×105/dish onto 10-cm culture dishes in triplicate. Cells were transduced with 4.4×107 pfu of either Ad-CMV or Ad-CMV-RB2/p130 and counted every 24 hours for 6 days. Cells were also collected, and Western blot analysis was performed essentially as previously described.9 The anti-pRb2/p130 was used at a dilution of 1:1000; the anti–β-tubulin (Sigma Chemical Co) was used at a dilution of 1:1000; and the E2F-4 and E2F-5 antibodies (Santa Cruz Biochemicals) were used at a dilution of 1:500.
Dose-Response Growth Curve and Flow Cytometry Analysis (FACS)
PASM cells were plated at a density of 1×105/dish onto 10-cm culture dishes in triplicate. Cells were transduced with 5, 10, or 50 multiplicity of infection (MOI) of Ad-CMV, Ad-CMV-β-Gal, or Ad-CMV-RB2/p130. The cells were counted every 24 hours for 5 days. FACS analysis was performed as previously described.9
Gel Shift Assay (Electrophoretic Mobility Shift Assay)
Briefly, PASM cells were seeded at a density of 1×106/dish onto 10-cm culture dishes in duplicate. Cells were transduced with 1×108 pfu of Ad-CMV or Ad-CMV-RB2/p130. Cells were collected, and gel shift assays were performed essentially as previously described.17
The animal groups treated with Ad-CMV or Ad-RB2/p130 consisted of 10 rats each, whereas the group infused with Ad-β-Gal consisted of 5 rats. The technique of endothelial denudation was set up for these three groups and for a control group of 10 untreated rats as previously determined.18 19 After balloon injury, a segment of common artery ≈1 cm long was isolated through the placement of vascular clamps on the proximal common and proximal internal arteries. For each rat, a total of 50 μL of 1×107 pfu/μL adenoviral vector was instilled into the isolated common carotid segment. After 20 minutes of incubation, the vector-containing medium was withdrawn, the external carotid artery (CA) was ligated, and the blood flow through the common and internal carotid arteries was reestablished.
Two weeks after gene transduction, the carotid arteries were dissected free from the surrounding tissues, and the rats were euthanized. Structural, ultrastructural, and immunohistochemical analyses were performed as previously described.18 19
Blood vessel wall thickness and lumen diameter were evaluated with the use of a Digital Imaging Processing (Image-Pro Plus; Media Cybernetics).
Statistical analysis was performed as previously described.19 A value of P<0.05 was considered statistically significant.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
Effects of Viral Transduction of the RB2/p130 Gene on the Growth of a PASM Cell Line
The PASM cell line expresses many differentiation markers of SMCs and can therefore be considered a good paradigm.14 20 PASM cells were plated at a density of 1×105 onto 10-cm culture dishes in triplicate and transduced with 50 pfu/cell of Ad-CMV or Ad-CMV-RB2/p130.
Cells were counted according to the trypan blue exclusion method each day during 1 week to monitor their growth rate. In PASM cells transduced with Ad-CMV-RB2/p130, the growth rate was inhibited by 4-fold compared with that of Ad-CMV–transduced cells (Figure 1A⇓). To measure the expression level of pRb2/p130 in Ad-CMV-RB2/p130– and Ad-CMV–transduced cells, we harvested cells each day for 1 week and performed Western blot analysis on the extracts. At each time point, the expression of pRb2/p130 was ≈200-fold higher in the Ad-CMV-RB2/p130– than in the Ad-CMV–transduced cells (Figure 1B⇓). The blot was normalized for equal loading and transfer of proteins by blotting the membrane with anti–β-tubulin (Figure 1B⇓) and by staining the membrane with Coomassie Brilliant Blue (data not shown). The cell count in uninfected PASM cells was comparable to that of Ad-CMV–infected cells.
Effects of Different Amounts of Adenoviral Particles on the Growth of a PASM Cell Line
PASM cells were plated at a density of 1×105 onto 10-cm culture dishes in triplicate and transduced with 5, 10, or 50 pfu/cell of Ad-CMV, Ad-CMV-β-Gal, or Ad-CMV-RB2/p130.
Cells were counted according to the trypan blue exclusion method each day during 5 days to monitor their growth rate. In PASM cells transduced with 10 or 50 MOI of Ad-CMV-RB2/p130, the growth rate was inhibited by almost 4-fold compared with that of Ad-CMV– and Ad-CMV-β-Gal–transduced cells (Figure 2⇓). The cell count in Ad-CMV–infected PASM cells was comparable to that of Ad-CMV-β-Gal–infected cells.
To test whether exposure of the cultured cells to adenovirus at 50 MOI could be associated with cytotoxicity, we transduced PASM cells with Ad-CMV, Ad-CMV-β-Gal, or Ad-CMV-RB2/p130 and 48 hours later processed the samples for FACS analysis. Figure 2⇑ shows that exposure of PASM cells to any of the 3 adenoviruses did not cause apparent cytotoxicity. In addition, as we previously demonstrated, overexpression of pRb2/p130 resulted in a G0 accumulation of the cells compared with the control (Ad-CMV and Ad-CMV-β-Gal) transduced cells (Figure 2⇑).9
pRb2/p130 Associates In Vivo With E2F Family Members and Inhibits Their Ability to Transactivate Genes That Promote Cell Cycle in PASM Cells
Because the growth-suppressive function of the retinoblastoma family of proteins is thought to occur at least in part through their binding and negative regulation of specific members of the E2F family of transcription factors, we analyzed the E2F complexes in PASM cells infected with Ad-CMV-RB2/p130 or Ad-CMV cells. In vivo, pRb2/p130 associates with E2F-4 and E2F-5 and thereby inhibits their ability to transactivate genes that promote cell cycle progression.21 PASM cells were plated at a density of 2×106 cells, transduced with either Ad-CMV or Ad-CMV-RB2/p130 at a concentration of 50 pfu/cell, and harvested after 48 hours. Through the use of electrophoretic mobility shift assay with an oligonucleotide probe of the E2F DNA binding sequence labeled with γ-32P-dCTP, we detected an E2F complex that effectively competed with a nonradiolabeled wild-type oligonucleotide but not with a point-mutated oligonucleotide that abrogates E2F binding to DNA (Figure 3A⇓ and 3B⇓, lanes 1 to 3 and 5 to 7). The band of the E2F complex was supershifted through incubation with an antibody that specifically recognizes pRb2/p130 (Figure 3A⇓, lanes 4 and 8, arrow on the left), as well as by an antibody that specifically recognizes E2F-4 (Figure 3B⇓, lanes 4 and 8, arrow on the left), in both the Ad-CMV-Rb2/p130– and Ad-CMV–transduced cells. Almost the entire E2F complex was shifted in the Ad-CMV-RB2/p130–infected cells through incubation of the pRb2/p130 antibody (Figure 3A⇓, lane 8), indicating that most of the E2F is bound by pRb2/p130 in these cells. However, in the Ad-CMV–transduced cells, only a small fraction of the E2F complex was supershifted by the pRb2/p130 antibody (Figure 3A⇓, lane 4), a reflection of the low endogenous expression level of pRb2/p130 in the proliferating PASM cells (Figure 1B⇑). This suggests that the transduction of SMCs by Ad-CMV-RB2/p130 and the resulting high level of expression of pRb2/p130 provide an abundance of pRb2/p130 that can effectively sequester E2F activity, thereby leading to growth arrest. In addition, the band of the E2F complex was supershifted through incubation with an antibody that recognize E2F-4 (Figure 3B⇓, lanes 4 and 8, arrow on the left). However, because in the Ad-RB2/p130–transduced cells a smaller fraction of the E2F complex was supershifted by the E2F-4 antibody (Figure 3B⇓, lane 8), we decided to analyze the expression of the E2F family members after transduction. As shown in Figure 4⇓, the expression level of E2F-5 was not affected by the adenovirus carrying RB2/p130 or the adenovirus control. The expression of E2F-4 was instead down-regulated on overexpression of pRb2/p130, suggesting that its overexpression in PASM cells could somehow target E2F-4 for protein instability and degradation or could inhibit its transcription.
The effects of RB2/p130 transduction on neointimal formation at 2 weeks after balloon injury were analyzed. In the animal group in which we infused Ad-CMV, we observed a reproducible neointimal formation as shown by semithin sections stained with methylene blue (Figure 5a⇓). We found an increase in the intimal thickness for the presence of several layers of SMCs with the interruption of the inner elastic lamina, as shown in Figure 5a⇓ (top and bottom insets, respectively). This resulted in a great decrease in the lumen (276.4±25.4 μm) and, conversely, an increase in the thickness of the medial and intimal carotid layer (308±74 μm) compared with the left CA (lumen 758.8±41.85 μm, thickness 57.4±1.2 μm), as also shown in Figure 6A⇓ and 6B⇓, respectively. On electron microscopy, the neointima of the rats treated with Ad-CMV appeared almost to be entirely constituted of layers of SMCs immersed into an intercellular matrix that included reticular fibrils, without any trace of neovascularization (Figure 5b⇓ and 5c⇓). In the uninfected animal group, we observed a neointimal formation comparable to previously published work (data not shown).11 12
In the group treated with Ad-RB2/p130, neointimal formation was highly suppressed 14 days after injury. The lumen of the right carotid arteries perfused with Ad-RB2/p130 (lumen 712.38±117.36 μm) was comparable to that of the left uninjured side (lumen 652.63±87.23 μm) (Figure 6A⇑). There were essentially no differences between the thickness of the arterial walls treated with Ad-RB2/p130 (thickness 60.5±6.18 μm) and that of the normal uninjured left side (thickness 64.8±8.68 μm) (Figure 6B⇑). The differences between the animal group treated with Ad-CMV or Ad-RB2/p130 were found to be statistically significant (P<0.01). However, no statistically significant differences in the lumen size or arterial thickness were found between the right CA treated with Ad-RB2/p130 or the left untreated CA, indicating that RB2/p130 greatly inhibited the proliferative capacity of the SMCs in vivo as in vitro (Figures 1A⇑, 5⇑, and 6⇑). On electron microscopy, we were able to demonstrate that the intimal layer of the CAs treated with Ad-RB2/p130 consisted solely of endothelial cells (Figure 5b⇑ and 5c⇑). We compared the morphological aspects of the Ad-RB2/p130–treated CAs with the contralateral uninjured side (left). Interestingly, semithin cross sections of the left CAs (controls) stained with methylene blue and observed with light microscopy showed normal thickness of the arterial wall and typical endothelial cells limiting the lumen, as determined with electron microscopy (Figure 7a⇓ and 7b⇓). This is identical to the Ad-RB2/p130–treated CAs.
Finally, we investigated the expression levels of pRb2/p130 after transduction in vivo with the use of immunohistochemistry. Sections of CAs treated with Ad-RB2/p130 (Figure 8b⇓) showed a high level of pRb2/p130 compared with the left normal uninjured side (Figure 8a⇓), demonstrating in vivo adenoviral gene transfer of RB2/p130 and the constitutively high expression of the transduced gene after 2 weeks (Figure 8⇓). The efficacy of gene transfer is demonstrated by the high-power-field photomicrograph (×100) that identifies single cells positive for nuclear pRb2/p130 staining (Figure 8c⇓), as well as by the high expression level of β-galactosidase transgene in Ad-β-Gal–infected vessels (Figure 8d⇓). Figure 8e⇓ shows the high-power field (×100) of Figure 8d⇓.
The major finding of this study is that adenovirus-mediated RB2/p130 gene transfer can prevent SMC proliferation after balloon injury in vivo.
The retinoblastoma gene (RB) appears to be of central importance in the control of S-phase progression. In fact, the deletion or inactivation of both RB alleles induces malignant progression of a large number of tumors, and its functional restoration into either primary or malignant cell lines has been shown to inhibit cellular proliferation.22 The proliferation of SMCs is associated with the expression of a variety of growth factors and inflammatory cytokines, which stimulate SMC migration from the media into the intima, neointima formation, and, eventually, aggravation of atherosclerosis or induction of restenosis.23 It has been clearly demonstrated that many factors that regulate signal transduction and cell cycle progression may induce SMC proliferation in vitro and in vivo.2 This evidence led to the idea that critical regulatory proteins of the cell cycle machinery may offer the potential for inhibition of cell proliferation and perhaps of intimal thickening after arterial injury. The retinoblastoma family proteins (pRb/p105, p107, and pRb2/p130) are excellent candidates for gene therapy of vascular disease. In particular, pRb2/p130 has been shown to inhibit cellular proliferation both in vitro and in vivo.2 8 9 10 24 In the present study, we demonstrate that adenovirus-mediated RB2/p130 gene transduction greatly inhibited SMC proliferation in vitro and in vivo. Using the established model of rat carotid artery restenosis,11 12 we show that adenovirus-mediated localized arterial transduction of RB2/p130 at the time of PTCA drastically reduces neointimal hyperplasia and prevents restenosis. Moreover, the ability of pRB2/p130 to block proliferation correlated with its capacity to bind and sequester transcription factor regulators E2Fs. In addition, our data demonstrated that pRb2/p130 downregulates the E2F-4 protein level in SMCs, suggesting an effect on E2F-4 gene expression similar to the effects of pRb/105 on E2F-1.25
The vast majority of endogenous E2F activity during the G0/G1 transition stems from E2F-4.26 In fact, during the G1 phase of the cell cycle, E2F-4 accounts for almost all of the free activity.26 During the S phase, an equal mixture of E2F-4 and E2F-1 composes the free E2F activity.27 The main form of E2F detected in the G0/G1 phases in primary mouse fibroblasts is E2F-4 bound to pRb2/p130, which is then replaced by p107/E2F-4 complexes in late G1.26 27 Therefore, pRb2/p130 is the major negative modulator of E2F activity in quiescent or resting cells and offers the potential for inhibition of cellular proliferation and intimal thickening after balloon angioplasty. Together, these observations strongly indicate that RB2/p130 could be a critical molecule for vascular gene therapy.
This work was supported by “Fondi 1% SSN” (Progetto Terapia Genica Cardiovascolare and Progetto Finalizzato IRCCS), AIRC, institutional funds from Thomas Jefferson University (Dr Condorelli), and by Telethon E.735 (Dr Stassi). This work was also supported by NIH Grants RO1-CA-60999-01A1 and PO1-NS-36466 and by the Sbarro Institute for Cancer Research and Molecular Medicine (Dr Giordano). Dr Claudio is the recipient of a fellowship from the Associazione Leonardo di Capua. Dr Condorelli would like to thank Dr Carlo M. Croce for his support and encouragement.
- Received May 26, 1999.
- Accepted September 14, 1999.
- © 1999 American Heart Association, Inc.
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