(Hypertension. 1999;34:192-200.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
Inhibition of the p53 Tumor Suppressor Gene Results in Growth of Human Aortic Vascular Smooth Muscle Cells
Potential Role of p53 in Regulation of Vascular Smooth Muscle Cell Growth
From the Department of Geriatric Medicine (M.A., H.M., S.H., H.N., K.Y., A.M., J.H., T.O.) and Division of Gene Therapy Science (R.M., Y.K.), Osaka University Medical School, Suita, Japan.
Correspondence to Ryuichi Morishita, MD, PhD, Division of Gene Therapy Science, Osaka University Medical School, 2-2 Yamada-oka, Suita 565, Japan.
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Abstract—Loss of activity of the p53 tumor suppressor gene product has been postulated in the pathogenesis of human restenosis. Although the antioncogenes p53 and retinoblastoma (Rb) susceptibility gene have been reported to play a pivotal role in cell cycle progression in various cells, the role of p53 and Rb in the growth of human vascular smooth muscle cells (VSMC) has not yet been clarified. We used antisense strategy against p53 and Rb genes by the viral envelope–liposomal method. Transfection of antisense p53 oligodeoxynucleotides (ODN) alone resulted in an increase in DNA synthesis compared with control (P<0.01). Similarly, transfection of antisense Rb ODN alone resulted in a higher DNA synthesis rate than control (P<0.01). Moreover, increase in VSMC number was only induced by transfection of antisense p53 ODN alone or cotransfection of p53/Rb ODN (P<0.01), whereas a single transfection of antisense Rb ODN had little effect on cell number. Therefore, we hypothesized that this discrepancy is due to the induction of apoptosis mediated by p53. Interestingly, apoptotic cells were markedly increased in VSMC transfected with antisense Rb ODN alone, accompanied by the induction of p53 protein. The number of apoptotic cells was attenuated by cotransfection of antisense p53 ODN (P<0.01). We finally examined the molecular mechanisms of apoptosis induced by the absence of Rb. In VSMC transfected with antisense Rb ODN, bax, a promoter of apoptosis, was significantly increased in VSMC transfected with antisense Rb ODN (P<0.01), whereas bcl-2 and Fas did not play a pivotal role in the induction of apoptosis. Overall, these data first demonstrated that the antioncogenes p53 and Rb negatively regulated the cell cycle in VSMC, suggesting that the modulation of their activity may mediate VSMC growth such as that in restenosis and atherosclerosis. The presence of p53 plays a pivotal role in the regulation of apoptosis in human VSMC growth, probably through the bax pathway. These results provide evidence that p53 is a functional link between cell growth and apoptosis in VSMC.
Key Words: antisense • genes • apoptosis • restenosis • muscle, smooth, vascular
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Introduction |
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Intimal hyperplasia is the pathological process that underlies restenosis, atherosclerosis, and vascular graft occlusion; it develops in large part as a result of vascular smooth muscle cell (VSMC) proliferation and migration induced by a complex interaction of multiple growth factors that are activated by vascular injury.1 The process of VSMC proliferation is dependent on the coordinated activation of a series of cell cycle regulatory genes that results in mitosis. A critical element of cell cycle progression regulation involves the complex formed by E2F, cyclin A, and cdk 2.2 The dissociation of the transcription factor E2F from this complex is proposed to play a pivotal role in the regulation of cell proliferation by inducing the coordinated transactivation of genes involved in cell cycle regulation. The importance of cell cycle regulation is apparently great, because we and others have previously reported the successful prevention of restenosis after angioplasty with antisense oligodeoxynucleotides (ODN) against cell cycle regulatory genes, decoy cis element of E2F binding site, and gene transfer of nonphosphorylated Rb (retinoblastoma gene).3 4 5 6 Therefore, research has focused on the role of antioncogenes in the regulation of VSMC growth. In particular, p53 (p53 tumor suppressor gene) and Rb have been postulated to negatively regulate the cell cycle in various cell types.7 8 9 However, little is known about the role of p53 and Rb in the regulation of VSMC. The presence of a functional p53 protein has been implicated as a critical determinant to regulate DNA replication, DNA repair, and programmed cell death.7 9 10 11 On the other hand, the antiproliferative effect of the Rb product has also been reported to depend on its capacity to bind to E2F and thereby prevent this transcription factor from binding to the E2F cis element within the promoters of the essential cell cycle regulatory genes.7 8 9 10 Therefore, the abnormal progression of the cell cycle seen in cancer is thought to result from the mutation of these negative cell cycle regulatory genes, especially p53 and Rb.7 8 9 Of importance, recent studies suggested that loss of p53 activity may be responsible for the pathogenesis of human restenosis.12 13 14 From this viewpoint, it is necessary to understand the negative regulation of the cell cycle by p53 and Rb in human VSMC. This study examines the role of p53 and Rb in negative regulation of the cell cycle in human VSMC with the use of antisense strategy.
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Cell Culture
Human aortic VSMC (passage 5) were obtained from Clonetics Corp (San Diego, Calif) and cultured in modified MCDB131 medium supplemented with 5% fetal calf serum, 100 U/mL penicillin, 100 mg/mL streptomycin, 10 ng/mL epidermal growth factor, 2 ng/mL basic fibroblast growth factor, and 1 mmol/L dexamethasone in the standard fashion.15 All the cells were used within passages 5 to 6.
Synthesis of ODN and Selection of Target Sequences
The sequences of phosphorothioate ODN against human p53 and Rb
were as previously reported16 17 : (Rb antisense:
5'-GTG-AAC-GAC-ATC-TCA-TCT-AGG-3'; Rb sense:
5'-CCT-AGA-TGA-GAT-GTC-GGT-CAC-3'; p53 antisense:
5'-CGG-CTC-CTC-CAT-GGC-AGT-3'; p53 sense:
5'-ACT-GCC-ATG-GAG-GAG-CCG-3'; scrambled Rb:
5'-AGC-TAG-CTA-GCT-AGC-TAG-CTA-3'; scrambled p53:
5'-AGT-GGC-CTG-CAT-CTC-CGC-3'; antisense thrombomodulin:
5'-ACC-CAG-AAA-GAA-AAT-CCC-3'. These antisense ODN inhibit human p53
and Rb synthesis in human hematopoietic cells.16 17
We also used scrambled ODN (5'-CGT-CGT-CGG-TAC-CGT-CCA-3') as negative
control.
Preparation of Hemagglutinating Virus of Japan
Liposomes
Phosphatidylserine, phosphatidylcholine, and
cholesterol were mixed in a weight ratio of
1:4.8:2.3 4 5 Dried lipid was hydrated in 200 µL balanced
salt solution (BSS) (137 mmol/L NaCl, 5.4 mmol/L KCl, 10
mmol/L Tris-HCl, pH 7.6) containing sense or antisense ODN. The control
liposome complex contained BSS without ODN. Purified hemagglutinating
virus of Japan (HVJ) (Z strain) was inactivated by UV
irradiation (110 ergs/mm2 per second) for 3
minutes just before use. The liposome suspension was mixed with HVJ
(20 000 hemagglutinating units). The mixture was incubated at 4°C
for 10 minutes and then for 60 minutes with gentle shaking at 37°C.
Free HVJ was removed from the HVJ liposomes by sucrose density gradient
centrifugation. The top layer of the sucrose gradient
was collected for use.
Effect of Antisense ODN on DNA Synthesis
VSMC were seeded onto 96-well tissue culture plates. At 80%
confluence, VSMC were rendered quiescent by incubation for 48 hours in
defined serum-free medium (DSF) supplemented with insulin
(5x10-7 mol/L), transferrin (5 mg/mL), and
ascorbate (0.2 mmol/L).18 Then 10 µL HVJ liposomes
(containing 15 µmol/L ODN) was added to the wells. The cells
were incubated at 4°C for 10 minutes and then at 37°C for 30
minutes. The cells were washed 3 times with BSS containing 2
mmol/L CaCl2 and incubated in DSF for 16 hours.
Relative rates of DNA synthesis were assessed by determination of
3H-thymidine incorporation into trichloroacetic
acid–precipitable material over the next 24 hours after 16 hours of
incubation.19
Counting of Cell Number
Human aortic VSMC were seeded onto uncoated 96-well tissue
culture plates (Corning). After cells reached 80% confluence, the
medium was changed to fresh DSF. The cells were then incubated for 48
hours. Then transfection of ODN was performed as described above. On
day 1, the medium was again changed to fresh DSF containing 0.05%
fetal bovine serum (GIBCO). After 3 days, an index of cell
proliferation was determined with a water soluble tetrazolium (WST)
cell counting kit (Wako).20 Briefly, 50 000 cells per
well reflects absorbance of 1 for the manufacturer's recommended
conditions. The sensitivity of the WST assay is double that of the
3-(4,5-dimethyl-thiazole-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)
assay. For our experimental conditions, an increase in absorbance of
0.2 reflects increase in cell number from 20 000 cells per
well.
Counting of Apoptotic Cells
As an assay of cell death by apoptosis, we used
fluorescent DNA-binding dyes to define nuclear chromatin
morphological features as a quantitative index of apoptosis
within the cell culture system. Cells to be analyzed for
apoptosis were stained with Hoechst 33342 and propidium
iodide and viewed under fluorescence microscopy as previously
described.21 22 23 24 The use of both membrane-permeable
(H33342) and -impermeable (PI) dyes in the assay allowed the
determination of cell viability and plasma membrane integrity and an
accounting of any nonapoptotic toxic or necrotic death induced
in the study groups. Cells were seeded onto 6-well dishes
(Laboratory-Tek) and were cultured in DSF for 2 days after
subconfluence. Transfection procedures were as described above.
To stain the cells for DNA, they were incubated with Hoechst 33342 (5
µg/mL in PBS) for 20 minutes at 37°C.23 Individual
nuclei were visualized at x400 to distinguish the normal uniform
nuclear pattern from the characteristic condensed coalesced chromatin
pattern of apoptotic cells. To quantify apoptosis, 400
nuclei from random microscopic fields were analyzed by an
observer blinded to the treatment groups.
In addition, we used the measurement of cellular DNA fragmentation with a cellular DNA fragmentation ELISA kit (Boehringer Mannheim) to quantity apoptosis induced by antisense ODN.25 Briefly, measurement of 10 000 apoptotic cells per well reflects absorbance of 1.5 for the manufacturer's recommended conditions. The sensitivity of DNA fragmentation ELISA assay is well correlated with the results from the conventional 3H-thymidine–based DNA fragmentation assay. For our experimental conditions, increase in absorbance of 0.2 reflects increase in cell number from 2000 apoptotic cells per well.
Western Blot
Western blot was performed for analysis of Rb, p53, bax,
and bcl-2 proteins. VSMC were seeded onto 10-cm dishes. VSMC were grown
to confluence and made quiescent by incubation in DSF before
transfection. Seventy-two hours after transfection, the cells were
fixed with 10% trichloroacetic acid in saline, followed by extraction
of total protein with urea-TX (9 mol/L urea, 2% Triton-X, and 5%
2-mercaptoethanol). Samples containing 100 µg protein were run on
7.5% (Rb) or 12.5% (p53, bax, and bcl-2) sodium dodecyl
sulfate polyacrylamide gels. Proteins were separated by
SDS-PAGE, transferred to nitrocellulose membrane (Hybond ECL,
Amersham), and incubated with a monoclonal antibody to Rb (1:500;
Pharmingen), p53 (1:20; Calbiochem), bax (1:100; Calbiochem), or bcl-2
(1:100; DAKO) at 4°C overnight. To quantify and compare levels of
proteins, the density of each band was measured by densitometry
(Shimazu). Amounts of loaded proteins were equal, as confirmed by the
staining with Coomassie brilliant blue R (Sigma). Staining with
Coomassie brilliant blue R revealed identical protein amounts in all
samples of Western blotting. Western blotting of tubulin with the use
of anti-tubulin antibody (anti-human mouse IgG; 1:100; Oncogene) was
also performed to confirm the equal amounts of loaded proteins.
Flow Cytometry
For the detection of proliferating cell nuclear antigen
(PCNA) expression by flow cytometry, cells were first fixed at
-10°C for 5 minutes in
paraformaldehyde-lysine-periodate fixation solution 2
days after transfection. After removal from the fixation solution,
cells were washed in PBS and incubated with PCNA monoclonal antibody
for 30 minutes at 4°C, washed in PBS, and incubated with
fluorescent isothiocyanate–conjugated rabbit anti-mouse IgG
monoclonal antibody (DAKO, High Wycombe, England) for 20 minutes at
4°C. An irrelevant IgG1 monoclonal antibody was used in parallel as
an isotopic control (Oncogene Science, Cambridge, England) before
incubation with the conjugated secondary antibody. Flow cytometric
analysis was performed on a FACScan flow cytometer. The
assessment of cellular DNA content was also made with a flow cytometer.
The cell cycle distribution data were obtained with the use of the
Rectangle-Fit (R-FIT) mathematical algorithm of the FACScan/Cellfit
software program in the standard manner. Expression of Fas in human
aortic VSMC was also examined by flow cytometry with the use of
anti-Fas antibody (anti-mouse IgM; Tago).
Statistical Analysis
All values are expressed as mean±SEM. ANOVA with subsequent
Scheffé's test was used to determine the significance of
differences in multiple comparisons. P<0.05 was considered
significant.
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Effect of Antisense p53 and Rb ODN in Human VSMC
Initially, we examined the presence of products of p53 and Rb genes in human aortic VSMC. As shown in Figures 1 and 2, the presence of p53 and Rb protein in human aortic VSMC was confirmed by Western blot. Therefore, we tested whether transfection of antisense p53 and Rb ODN into VSMC has effects on human VSMC growth. Transfection of either antisense p53 ODN alone or antisense Rb ODN alone or cotransfection of antisense p53 and Rb ODN (p53/Rb ODN) resulted in a significant increase in DNA synthesis assessed by 3H-labeled thymidine incorporation (Table). Increase in DNA synthesis in VSMC transfected with antisense p53 ODN alone and p53/Rb ODN was significantly higher than that with antisense Rb ODN alone (P<0.01). In contrast, transfection of scrambled ODN did not alter DNA synthesis (data not shown). The specificity of antisense p53 ODN was confirmed by the observation that the marked decrease in p53 protein was only observed by transfection of antisense p53 ODN but not sense p53, scrambled p53, sense Rb, scrambled Rb, the combination of sense p53 and sense Rb ODN, and thrombomodulin antisense ODN (Figure 3). Marked decrease in p53 mRNA was also observed by the transfection of antisense p53 ODN but not sense p53 ODN, scrambled p53, sense Rb ODN, and antisense thrombomodulin ODN as assessed by Northern blotting (data not shown). The specificity of antisense Rb ODN was also confirmed by the observation that transfection of antisense Rb ODN into quiescent VSMC resulted in a decreased level of Rb protein (Figure 2). In contrast, transfection of sense Rb ODN, transfection of scrambled Rb ODN, cotransfection of sense p53 and sense Rb ODN, and transfection of antisense thrombomodulin ODN did not affect the level of Rb protein (Figures 2 and 4). It is noteworthy that in VSMC transfected with antisense Rb ODN, p53 protein was significantly increased compared with VSMC transfected with sense ODN (Figure 1). Protein of p21 (WAF1/SDI1) that was induced by p53 gene was also decreased in VSMC transfected with antisense p53 ODN (sense p53, 100%; scrambled p53, 104±2%; antisense p53 ODN, 63±5%; P<0.01 for antisense p53 ODN versus sense p53 and scrambled p53 ODN).
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Next, we examined the number of VSMC to investigate whether an increase in DNA synthesis stimulates cell growth. Of importance, transfection of antisense p53 ODN and antisense p53/Rb ODN resulted in a significant increase in number of VSMC compared with respective sense ODN–transfected VSMC, whereas there was no significant difference in VSMC number between VSMC transfected with antisense Rb ODN and sense Rb ODN (Table). In contrast, there was no significant change in number of VSMC and DNA synthesis transfected with sense p53 ODN, scrambled p53 ODN, sense Rb ODN, scrambled Rb ODN, sense p53/Rb ODN, or antisense thrombomodulin ODN. Moreover, the stimulatory effects of antisense p53 ODN on VSMC growth occurred in a dose-dependent manner, as shown in Figure 5. In contrast, transfection of neither sense p53 ODN, scrambled p53 ODN, nor antisense thrombomodulin ODN stimulated VSMC growth (data not shown).
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To confirm that antisense p53 and Rb ODN promoted cell cycle progression of VSMC, the cell cycle of VSMC transfected with antisense ODN was also analyzed by flow cytometry. PCNA-positive VSMC, as a marker of entry into the S phase, were markedly increased in cells transfected with antisense p53 ODN or antisense Rb ODN compared with sense ODN–transfected cells (Figure 6b), consistent with results on DNA synthesis. Transfection of sense p53 ODN, scrambled p53 ODN, sense Rb ODN, and antisense thrombomodulin ODN did not alter the cell cycle. As shown in Figure 6c, progression of the cell cycle into S and G2/M phases was also confirmed in VSMC transfected with antisense p53 ODN or antisense Rb ODN. These results demonstrated that transfection of either antisense p53 ODN or antisense Rb ODN resulted in cell cycle progression into the G2/M phase. However, there is a discrepancy between the increase in DNA synthesis and absence of increase in VSMC number in cells transfected with antisense Rb ODN alone. Cell cycle progression by antisense p53 ODN was also supported by the phosphorylation of Rb. As shown in Figure 7, phosphorylated Rb was clearly observed in VSMC transfected with antisense p53 ODN, whereas this occurred with neither sense p53 ODN nor scrambled p53 ODN.
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Apoptosis in VSMC Transfected With Antisense Rb
ODN
To investigate the mechanisms of this discrepancy, we have focused
on the role of p53, because p53 has been postulated to regulate
programmed cell death, as discussed earlier. Given the cell cycle
progression into the G2/M phase by transfection
of antisense Rb ODN alone, it is not likely that the lack of increase
in the number of VSMC lacking Rb ODN is due to the cell cycle
progression into the S phase but not to the G2/M
phase. Therefore, we hypothesized that the presence of p53 mediated
programmed cell death in VSMC transfected with antisense Rb ODN alone.
As shown in Figure 8a to 8c, the
number of apoptotic cells assessed by nuclear staining was
significantly increased by transfection of antisense Rb ODN alone
compared with sense ODN. Of importance, cotransfection of antisense p53
and Rb ODN attenuated the increase in number of apoptotic cells
(Figure 8d). In contrast,
transfection of sense p53 ODN, scrambled p53 ODN, and sense Rb ODN did
not increase the apoptotic cells (sense p53, 6.7±1.5%;
scrambled p53, 6.2±1.4%; sense Rb, 6.3±1.2%; antisense Rb,
21.7±5.2%; P<0.01 for antisense Rb versus other groups).
These results demonstrated the presence of p53-induced
apoptosis in VSMC lacking Rb protein by antisense Rb ODN. These
results were confirmed by the measurement of DNA fragmentation (Figure 9). Consistent with nuclear staining, DNA fragmentation
in VSMC transfected with antisense Rb ODN was significantly increased
compared with that in VSMC transfected with sense ODN in a
time-dependent manner (P<0.01). Increased DNA fragmentation
was also significantly attenuated by cotransfection of antisense p53
ODN (P<0.05). The specificity of apoptosis induced
by antisense Rb ODN was also supported by the observation that there
was no significant difference in DNA fragmentation rate at 4 days after
transfection among VSMC transfected with sense p53 ODN, scrambled p53
ODN, and sense Rb ODN (sense p53, 0.280±0.016; scrambled p53,
0.295±0.016; sense Rb, 0.276±0.017; antisense Rb, 0.428±0.038
absorbance; P<0.01 for antisense Rb versus other
groups).
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Molecular Mechanisms of Apoptosis in VSMC Transfected With
Antisense Rb ODN
Because p53 regulates apoptosis in bcl-2,
antiapoptotic gene, dependent pathway, and independent pathway,
levels of bcl-2 and bax were measured by Western blot. As shown
in Figure 10, bax protein was
significantly increased in VSMC transfected with antisense Rb ODN
compared with sense Rb ODN (P<0.01). More importantly,
upregulation of bax induced by transfection of Rb antisense ODN was
significantly abolished by cotransfection of p53 antisense ODN. In
contrast, as shown in Figure 11, transfection of antisense Rb ODN alone into VSMC also resulted in an
increase in bcl-2 protein compared with sense Rb ODN. The ratio of bax
to bcl-2 was significantly increased in VSMC transfected with antisense
Rb ODN (P<0.01, Figure 12),
possibly leading to apoptosis. Of importance,
consistent with the increased number of apoptotic VSMC,
the increase in the ratio of bax to bcl-2 was significantly attenuated
by cotransfection with antisense p53 ODN, in addition to antisense Rb
ODN (P<0.05). We also measured the expression of Fas, a
death factor, in VSMC treated with antisense Rb ODN. However,
transfection of antisense Rb ODN did not affect Fas expression compared
with sense Rb ODN, as assessed by flow cytometry (data not shown).
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A fundamental pathological feature of vascular disease is marked by the abnormal accumulation of cells within the intimal space, resulting in neointimal lesion formation produced by alterations in the homeostatic balance between cell growth and cell death.1 One successful approach is to target components of the common pathways that are shared by many growth factors. On the basis of this concept, we have reported that local delivery of antisense ODN directed against cell cycle regulatory genes and ODN containing the E2F cis element sequence as "decoys" inhibited neointimal formation in several models of vascular lesion formation.3 4 5 6 These results emphasize the importance of cell cycle regulation in the disease process. In contrast, negative regulation of the cell cycle is thought to be controlled by antioncogenes in many cell types. Because p53 and Rb have been reported to play a pivotal role in the regulation of cell growth,2 7 8 frequent mutations in these genes cause a break of cell cycle control, resulting in the neoplastic transformation in human cancers. Given that loss of p53 activity induced by cytomegaloviral infection might be related to the pathogenesis of restenosis,12 13 14 it is important to understand the negative cell cycle regulation in human VSMC. Therefore, we used a "loss of function" approach, with antisense ODN, to clarify the roles of p53 and Rb.
The present results showed that single transfection of antisense p53 ODN and cotransfection of p53/Rb ODN promoted DNA synthesis and growth of VSMC, whereas single transfection of antisense Rb ODN resulted in increased DNA synthesis but not number of VSMC (Table). The specificity of antisense ODN was supported by several observations: (1) the decrease in p53 or Rb protein level only by antisense p53 or antisense Rb ODN (Figures 1, 3, 4, and 5); (2) the decrease in p53 mRNA by antisense p53 ODN (Figure 2); (3) the decrease in p21 protein, an inducible protein by p53, by antisense p53 ODN; (4) the lack of response to either sense or scrambled ODN on DNA synthesis (Table); and (5) the lack of response to either sense or scrambled ODN on cell number (Table). Although we thought that transfection of antisense Rb ODN alone promoted cell cycle progression into the S phase but not the G2/M phase, our initial hypothesis was not supported by flow cytometry, which demonstrated that transfection of either antisense p53 ODN alone or antisense Rb ODN alone promoted cell cycle progression into both S and G2/M phases (Figure 6). Although the present study demonstrated negative control of the cell cycle by Rb in human VSMC, loss of Rb function is not enough to stimulate human VSMC growth. In contrast, loss of p53 activity in human aortic VSMC is sufficient to promote cell growth.
Because regulation of the intimal cell population requires a delicate balance between cell influx, cell growth, and cell death,1 we hypothesized that cells lacking Rb undergo cell death/apoptosis, resulting in no change in net balance of cell number despite cell cycle progression. It has become increasingly clear that the process of cell death by apoptosis is a relatively ubiquitous phenomenon observed in a variety of cell types, including VSMC.26 27 28 29 Indeed, recent studies suggest that apoptosis occurs within the context of atherosclerosis and restenosis after angioplasty.27 28 29 These phenomena were also confirmed in experimental animals, demonstrating that cell death by apoptosis appears to occur during the processes of vascular remodeling and lesion formation.30 31 Therefore, p53-mediated apoptosis demonstrated in this study is important in understanding the regulation of VSMC growth, because p53 is known to regulate DNA replication, DNA repair, and programmed cell death.7 9 10 The evidence of apoptosis induced by p53 in cells lacking Rb was supported by several observations: (1) the increase in apoptotic cells by antisense Rb ODN alone (Figure 8); (2) the increase in DNA fragmentation by antisense Rb ODN alone (Figure 9); and (3) the increase in number of apoptotic cells and DNA fragmentation that was abolished by cotransfection of antisense p53 ODN (Figures 8 and 9). The present data were consistent with the previous observation by Bennett et al32 that both disruption of Rb/E2F and inhibition of p53 are required for plaque VSMC to proliferate without apoptosis. In this study, we used antisense strategy, whereas Bennett et al used retroviral constructs expressing dominant-negative Rb and p53 genes to explore the interplay of these pathways. Moreover, our finding that p53 was upregulated in cells lacking Rb supports the previous finding that the ablation of Rb enhances apoptosis and overexpression suppresses apoptosis.32 Consistently, abnormally extensive apoptosis was detected in Rb-deficient mice.33 On the other hand, overexpression of E2F-1 gene resulted in escape from G0 into S phase, yet it also initiated p53-dependent apoptosis.34 Probably the inappropriate entry into the S phase, such as through loss of Rb and release of E2F, in the absence of survival factors may be associated with the activation of apoptosis in the presence of functional p53. In contrast, no increase in VSMC proliferation has been observed in p53 and Rb knockout mice.33 The findings from knockout mice seem to be different from those of the present study. This discrepancy between knockout mice and the present study is probably due to counterregulatory mechanisms during the developmental stage.
One of the functions of p53 is to repress expression of bcl-2 gene, which exhibits an antiapoptotic action through a cis-acting p53 negative-response element located in the 5'-untranslated region.35 However, apoptosis in VSMC transfected by antisense Rb ODN observed in this study is unlikely through the bcl-2 pathway, because upregulation of bcl-2 protein was observed in cells lacking Rb. Recently, p53 has also been reported as a direct transcriptional activator of bax gene, which is a homologous protein of bcl-2 gene but which attenuated bcl-2 function.36 Bcl-2 and bax are homologous proteins that have opposing effects on cell life and death, with bcl-2 serving to prolong cell survival and bax acting as an accelerator of apoptosis.37 The bcl-2 and bax proteins can form heterodimers in cells.37 Of importance, transfection of antisense Rb ODN resulted in a significant increase in bax expression in the presence of p53, leading to the significant increase in the ratio of bax to bcl-2 in VSMC that lack Rb. Apoptosis observed in this study may be mediated through the p53-bax pathway. The previous finding that VSMC from atherosclerotic plaques showed a higher number of apoptotic cells than normal VSMC despite the stable expression of bcl-2 gene suggests the presence of an independent apoptotic pathway from bcl-2 gene in human VSMC.22 Our data may explain the mechanisms of apoptosis observed in such atherosclerotic plaques and restenosis lesions. Consistent with our data, Bennett et al38 also reported that bcl-2 probably does not play a major role in regulation of apoptosis in VSMC. On the other hand, Fas, another death factor, may not be related to the apoptosis induced by antisense Rb ODN, because transfection of antisense Rb ODN did not affect Fas expression. Because it was previously reported that apoptosis of rat VSMC is regulated by p53-dependent and -independent pathways,38 other mechanisms might be involved in the apoptosis of VSMC. Furthermore, the sensitivity of apoptosis induced by p53 may be different among the species of VSMC (p53-sensitive human and rabbit VSMC and p53-resistant rat VSMC).39
Overall, these data first demonstrated that p53 and Rb negatively regulate the cell cycle in human aortic VSMC, suggesting that modulation of their activity may mediate VSMC growth such as that in restenosis after angioplasty and atherosclerosis. The product of p53, rather than Rb, may play a pivotal role in the regulation of apoptosis in abnormal human VSMC growth. Further studies of p53 and Rb in the pathological conditions in vascular disease may provide new insights into therapeutic strategies.
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Acknowledgments |
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This study was supported in part by grants from the Uehara Memorial Foundation, the Hoan-sya Foundation, the Japan Cardiovascular Research Foundation, a Japan Heart Foundation research grant, a grant-in-aid from the Tokyo Biochemical Research Foundation, and a grant-in-aid from the Ministry of Education, Science, Sports, and Culture. Dr Morishita is the recipient of a Harry Goldblatt Award from the Council of High Blood Pressure, American Heart Association. We wish to thank Chihiro Noguchi for excellent technical assistance.
Received February 9, 1999; first decision March 4, 1999; accepted April 6, 1999.
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