(Hypertension. 1998;31:493.)
© 1998 American Heart Association, Inc.
Scientific Contributions |
Inhibition of Growth of Human Vascular Smooth Muscle Cells by Overexpression of p21 Gene Through Induction of Apoptosis
From the Department of Geriatric Medicine, Osaka University Medical School, Suita, Japan (H.M., R.M., I.K., M.A., S.H., N.T., K.Y., A.M., J.H., T.O.), the Department of Radiation Biophysics, Kobe University, Kobe, Japan (A.N.), and the Institute for Cellular and Molecular Biology and Osaka University Medical School, Suita, Japan (Y.K.).
Correspondence to Toshio Ogihara, MD, PhD, Chief & Professor, Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamada-oka, Suita 565, Japan
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Abstract |
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The senescent cell-derived inhibitor (sdi)-1 protein (p21 product) has been identified as a downstream mediator of the tumor suppressor p53 in the regulation of cell cycle progression through a G1 phase checkpoint. Given the importance of cell cycle inhibition for the treatment of restenosis, in this study we focused on the function of p21 gene in inhibiting proliferation of vascular smooth muscle cells (VSMC). To test the hypothesis, we transfected human p21 gene into human aortic VSMC using hemagglutinating virus of Japan-liposome-mediated transfer. Initially, we examined the successful transfection of human p21 gene into VSMC. p21 protein was increased in VSMC transfected with p21 vector as compared with control vector. Accompanied by increased p21 protein, transfection of p21 vector resulted in a significant decrease in number of VSMC induced by 2% serum (P<.01). Although p21 has been reported to play an important role in the regulation of apoptosis in some cells, apoptosis mediated by p21 is still controversial. Therefore, we hypothesized that overexpression of p21 mediates apoptosis in human VSMC, in addition to the blockade of cell cycle progression. First, we assessed the concordance between morphologic analysis and apoptosis as determined by nuclear staining with Hoechst 33342. Cells transfected with p21 gene exhibited the characteristic features of cell shrinkage, membrane blebbing, and rounding that are typical of apoptotic death. Of greater interest, a significant increase in apoptotic cells was observed in VSMC transfected with p21 vector as compared with control vector (P<.01). These results were confirmed by the measurement of DNA fragmentation. Consistent with nuclear staining, DNA fragmentation in VSMC transfected with human p21 gene was significantly increased as compared with that in VSMC transfected with control vector (P<.05). To study the molecular mechanisms of apoptosis mediated by overexpression of p21 gene, the protein levels of bax, a promoter of apoptosis, and bcl-2, an inhibitor of apoptosis, were also measured by Western blotting. Overexpression of p21 gene significantly increased protein of bax (P<.05), whereas transfection of p21 gene did not alter bcl-2 protein. Importantly, the ratio of bax to bcl-2 was significantly increased in VSMC transfected with human p21 vector as compared with control vector (P<.05). Overall, these results demonstrated that inhibition of VSMC growth by overexpression of human p21 gene was accompanied by induction of apoptosis through an inappropriate increase in bax protein. These results suggest that regulation of cell cycle by p21 may be closely linked to programmed cell death/apoptosis in human VSMC.
Key Words: gene therapy • cell cycle regulatory gene • bax • bcl-2 • remodeling
Abbreviations: DSF = defined serum-free medium • HVJ = hemagglutinating virus of Japan • sdi-1 = senescent cell-derived inhibitor-1 • VSMC = vascular smooth muscle cells
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Introduction |
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Proliferation of vascular smooth muscle cells (VSMC) after arterial injury is important in the pathogenesis of a number of vascular proliferative disorders, including atherosclerosis and restenosis after balloon angioplasty. Neointimal hyperplasia after angioplasty, as well as vein grafting, involves many growth factors, including thrombin, fibroblast growth factor, and platelet-derived growth factor, which activate cell cycle progression.1–5 Since cyclin was first discovered, it has become clear that cell growth is dependent on the coordinated actions of cell cycle regulatory genes.6–9 We and others have reported previously that inhibition of the cell cycle resulted in blockade of neointimal formation in various balloon injury models.10–13 A growing number of observations in the field of vascular biology clearly demonstrate the importance of control of VSMC cell cycle progression by the retinoblastoma (Rb) gene product and tumor suppressor gene p53.6–9,14,15 More recently, p21, also known as sdi-1 (senescent cell-derived inhibitor), Cip1 (CDK-interacting protein), and WAF1 (wild-type p53-activated fragment), has been identified as a downstream mediator of the tumor suppressor p53 in the regulation of cell cycle progression through a G1 phase checkpoint.16–18 The efficacy of overexpression of p21 gene as gene therapy for restenosis has been reported.19–21 For example, adenoviral-mediated overexpression of human p21 inhibited restenosis in rat and porcine balloon injury models as well as in vitro VSMC growth by efficiently arresting VSMC in the G1 phase of the cell cycle.19,20 Similarly, our previous studies also demonstrated a gene therapy strategy using the p21 gene to prevent neointimal formation in a rabbit vein graft model by using hemagglutinating virus of Japan (HVJ)-liposomes.21 In addition to the blockade of cell cycle progression, p21 has also been reported to regulate apoptosis, DNA repair, and differentiation.16–18,22–27 However, postulated roles for p21 in apoptosis and DNA repair remain controversial. Studies suggest both induction and protection from apoptosis, as well as stimulatory and inhibitory effects of p21 in DNA repair.22–27 Moreover, few reports have mentioned the function of p21 in the regulation of VSMC growth. To elucidate the role of p21 in apoptosis in human aortic VSMC, we examined the effects of overexpression of human p21 gene on apoptosis in this study. We have addressed the following questions: 1) Can overexpression of p21 induce apoptosis in human VSMC, and 2) What is the molecular mechanism of apoptosis mediated by p21 in human VSMC?
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Materials and Methods |
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Construction of Plasmids
A plasmid expression vector containing human p21 gene driven by the SR
promoter was used in this study.16 This expression vector was derived from the plasmid pcDSR296 vector (donated by Dr. Yoshida, University of Tokyo, Japan).28 The control vector that did not contain p21 gene was used as a control plasmid in this study.
Preparation of HVJ-Liposomes
The preparation of the HVJ-liposome complex has been described previously.10,11,29,30 Briefly, phosphatidylserine, phosphatidylcholine, and cholesterol were mixed in a weight ratio of 1:4.8:2. The lipid mixture (10 mg) was deposited on the sides of a flask by removal of tetrahydrofuran in a rotary evaporator. Dried lipid was hydrated in 200 µL BSS (in mmol/L: 137 NaCl, 5.4 KCl, 13 Tris-HCl, pH 7.6) containing DNA-high mobility group (HMG)-1 complex (200 µg:64 µg), which had previously been incubated at 20°C for 1 hour. Liposomes were prepared by shaking and sonicating. Purified HVJ (Z strain) was inactivated by UV irradiation (110 erg/mm2/sec) for 3 minutes just before use. The liposome suspension (0.5 mL, containing 10 mg lipid) was mixed with HVJ (35,000 hemagglutinating units) in a total volume of 2 mL BSS. The mixture was incubated at 4°C for 10 minutes and then for 30 minutes with gentle shaking at 37°C. Free HVJ was separated from the HVJ-liposome complex by sucrose density gradient centrifugation. The top layer of the sucrose gradient was collected for use. We have previously reported that this preparation method of HVJ-liposome complex for transfection of plasmid DNA into VSMC is highly efficient.30,31
Cell Culture
Human aortic VSMC (passage 5) were obtained from Clonetics Corp. (San Diego, CA) 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 bovine fibroblast growth factor, and 1 mmol/L dexamethasone in the standard fashion. Cells were incubated at 37°C in a humidified atmosphere of 95% air/5% CO2 with changes of medium every 2 days. These cells showed the specific characteristics of VSMC, according to immunohistochemical examination and morphologic observation.32,33 Briefly, human aortic VSMC also tested positive for -actin and negative for expression of factor VIII antigen. All the cells were used within passage 5 to 7.
In Vitro Transfection of p21 Vector
VSMC (1x106) were inoculated on day 0 and grown to 80% confluence in 5% calf serum. After 80% confluence was reached, the medium was changed to fresh defined serum-free medium (DSF) containing 2% serum. DSF medium was supplemented with insulin (5x10-7 mol/L), transferrin (5 mg/mL), and ascorbate (0.2 mmol/ L).34 Then, cells were washed three times with BSS containing 2 mmol/L CaCl2.30,31 HVJ-liposome complex (500 µL) containing 1.3 mg lipid and 2.5 µg encapsulated DNA and HMG-1 was added to the dishes. The cells were incubated at 4°C for 5 minutes and then at 37°C for 30 minutes. After incubation, the medium was changed to fresh medium containing 2% serum, and cells were incubated overnight in a CO2 incubator. The medium was again replaced with fresh medium containing 2% serum. Cell counting and staining for apoptosis were performed on day 4 after transfection.
Western Blotting
VSMC were seeded onto 10-cm dishes. VSMC were grown to confluence and made quiescent by incubation in DSF medium, before transfection. At 4 days after transfection into VSMC, the cells were collected by centrifugation after scraping, followed by extraction of total protein with lysis buffer (50 mmol/L Tris-HCl, pH 8.0, 20 mmol/L El)TA, 1% SDS, 100 mmol/L NaCl). Samples containing 50 µg protein were run on 12.5% SDS-polyacrylamide gels. Proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane (Hybond ECL, Amersham, Buck-inghamshire, UK), and incubated with a monoclonal antibody to p21 (1:20, Calbiochem), bax (1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or bcl-2 (1:50; Santa Cruz) at 4°C overnight. Antibodies were diluted in 4% skimmed milk and 0.1% Tween-20 in PBS. The membranes were then washed and incubated with a 1:2000 dilution of mouse or rabbit Ig horseradish peroxidase-conjugated antibody (Amersham). Bound antibodies were detected by enhanced chemiluminescence (ECL, Amersham) and Hyperfilm-MP (Amersham). To quantify and compare levels of proteins, the density of each band was measured by densitometry (Shimazu, Kyoto, Japan). Amounts of loaded proteins were equal confirmed by the staining with CBB (Sigma Chemical Co., St. Louis, MO). Staining with CBB revealed the identical protein amounts in all samples of Western blotting (data not shown).
Counting of Cell Number
VSMC were seeded onto uncoated 96-well tissue culture plates (Corning, Corning, NY). In the preparation of experiments for determination of cell count, the cells were grown to subconfluence. The cells were then incubated in DSF for 48 hours. After transfection, the medium was again changed to fresh DSF with 2% serum. Fresh medium with 2% serum was changed every 48 hours. On day 4, an index of cell proliferation was determined using sulfonated tetrazolium salt, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) cell counting kit which is similar to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Wako, Osaka, Japan), because this compound produces a highly water-soluble formazan dye, which makes the assay procedure easier to perform.35 Tetrazolium salt has been used to develop a quantitative colorimetric assay for cell growth. The assay detects living, but not dead, cells. We confirmed that serum-stimulated increase in cell number was associated by increased absorbance at 450 nm (data not shown). Briefly, 50,000 cells/well reflects absorbance 1 in the manufacture’s recommended condition. The sensitivity of the WST assay is double that of the MTT assay. Under our experimental conditions, an increase in absorbance of 0.2 reflects an increase in cell number from 20,000 cells/well.
Counting of Apoptotic Cells
As an assay of cell death by apoptosis, we used fluorescent DNA-binding dyes to define nuclear chromatin morphologic features as a quantitative index of apoptosis within the cell culture system.36–38 Cells to be analyzed for apoptosis were stained with Hoechst 33342 and viewed under fluorescence microscopy.36–38 The use of membrane-permeable (H33342) dye allowed the determination of cell viability, plasma membrane integrity, and an accounting of any nonapoptotic toxic or necrotic death induced in the study groups. Cells were cultured in DSF medium for 2 days after reaching subconfluence. Transfection procedures were as described above. After staining with Hoechst 33342 (5 µg/mL in PBS), cells were counted under fluorescence microscopy.36–38 Individual nuclei were visualized at x400 to distinguish the normal uniform nuclear pattern from the characteristic condensed coalesced chromatin pattern of apoptotic cells. Although chromatin undergoes condensation during mitosis, these cells can be readily distinguished from apoptotic cells by their uniform and equatorial pattern of chromatin condensation compared with the randomly coalesced pattern typical of apoptotic cells. To quantify apoptosis, 400 nuclei from random microscopic fields were analyzed by an observer blinded to the treatment groups. The total number of apoptotic cells in each section was summed and expressed as a percentage of the total cell number. At least 10 individual sections were evaluated per slide. Samples were coded so that the analysis was performed without knowledge of which treatment each individual cells had received. The reproducibility of the results was reported previously (intraobserver variability 2.4±0.3%, interobserver variability 3.4±0.5%).36 Photographs were obtained with a fluorescence microscope (x200, x400) (Olympus). The apoptotic index obtained is a cross-sectional and not a cumulative assessment at the time of harvest and may be an underestimate of the total apoptotic population, because apoptotic cells in prenuclear condensation phase would be scored as normal and because late-stage apoptotic cells whose cellular membranes have disintegrated may not be detected.
Also, we employed the measurement of cellular DNA fragmentation using cellular DNA fragmentation ELISA kit (Boehringer Mannheim, Germany), to quantify apoptosis.39 Cultured VSMC were incubated with 10 µmol/L BrdU overnight at 37°C in 5% CO2. At 4 days after transfection, lysing solution containing BSA, EDTA, and Tween-20 was added to each well. DNA fragments in 100 µL cell lysate supernatant were tested by ELISA. The supernatant was transferred to an anti-DNA-precoated microtiter plate and incubated for 60 minutes at 37°C. After washing, the samples were denatured and fixed by microwave irradiation for 5 minutes. After cooling the microtiter plate for 10 minutes at -20°C, anti-BrdU peroxidase-conjugated solution was added and the plate was incubated for 60 minutes at 37°C. Wells were again washed, TMB substrate solution was added, and the plate was incubated for 30 minutes at room temperature. Stopping solution (25 µL of 1 mol/L H2SO4) was then added to each well. Absorbance was measured at 450 nm (reference wavelength 690 nm). We confirmed that apoptosis by hypoxia is associated by decreased absorbance. Briefly, 10,000 apoptotic cells/ well reflects absorbance 1.5 in the manufacturer’s recommended condition. The sensitivity of DNA fragmentation ELISA assay is well correlated with the results from the conventional [3H]thymidine-based DNA fragmentation assay. Under our experimental conditions, an increase in absorbance of 0.2 reflects an increase in cell number from 2,000 apoptotic cells/well.
Statistical Analysis
All values are expressed as mean±SEM. ANOVA with subsequent Bonferroni’s test was used to determine the significance of differences in multiple comparisons. Values of P<.05 were considered statistically significant.
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Results |
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First, we examined the transfection of human p21 gene into human VSMC assessed by Western blotting. As shown in Fig 1, successful transfer of human p21 gene by HVJ-liposome complex was confirmed by Western blotting of p21 protein. In p21 vector-transfected VSMC, the existence of p21 protein with identical molecular size to that previously reported was detected by Western blotting. In contrast, a weak band could be observed in control vector-transfected vessels. Consistent with the previous reports,16 transfection of human p21 gene resulted in significant inhibition of VSMC growth induced by serum (2%) stimulation as compared with control vector-transfected VSMC and untransfected VSMC (P<.01; Fig 2). Therefore, we hypothesized that overexpression of p21 mediated programmed cell death in VSMC. We counted the apoptotic cells in human aortic VSMC transfected with p21 gene or control vector. As shown in Fig 3A, we assessed the concordance between morphologic analysis and apoptosis determined by nuclear staining with Hoechst 33342. Under phase-contrast microscopy, some cells transfected with p21 gene exhibited the characteristic features of cell shrinkage, membrane blebbing, and rounding that are typical of apoptotic death. Simultaneous assessment of nuclear chromatic morphology by Hoechst 33342 staining verified that these cells eventually manifested typical apoptotic condensed and coalesced nuclei (Fig 3A). Interestingly, morphologic studies revealed a significant increase in apoptotic cells in VSMC transfected with p21 gene as compared to control vector (P<.01; Fig 3B). In untransfected VSMC, apoptotic cells were also very low in number, similar to VSMC transfected with control vector. These results were confirmed by the measurement of DNA fragmentation (Fig 4). Consistent with nuclear staining. DNA fragmentation in VSMC transfected with human p21 gene was modestly increased (
30%, P<.01 versus vehicle, P<.05 versus control) as compared with that in VSMC transfected with control vector. There was no significant change in DNA fragmentation rate between untransfected VSMC and VSMC transfected with control vector.
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Because bcl-2, an anti-apoptotic gene, and bax, a pro-apoptotic gene, are known to regulate apoptosis,40 levels of bcl-2 and bax were measured by Western blotting. As shown in Fig 5, bax protein was significantly increased in VSMC transfected with p21 gene as compared with control vector (P<.05). There was no significant difference in bax protein between untransfected VSMC and VSMC transfected with control vector. In contrast, as shown in Fig 6, transfection of p21 vector into VSMC did not alter bcl-2 protein as compared with control vector. Thus, the ratio of bax to bcl-2 was significantly increased in VSMC transfected with p21 vector as compared with untransfected VSMC and VSMC transfected with control vector (P<.05; Fig 7), possibly leading to apoptosis.
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Discussion |
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Recent studies have shown that p21 inhibits a complex of cdk kinases and cyclins, which plays a pivotal role in cell cycle progression.6–9 In addition to its ability to inhibit kinase activities of cyclin/CDK complexes, p21 has also been reported to bind to and to inhibit the activity of the DNA polymerase
cofactor PCNA.16–18,22 Thus, researchers postulate that the p21 gene may be an ideal target for overexpression in the restenosis model as well as the vein graft model. Indeed, in a balloon injury model, in vivo transfection of p21 gene resulted in the inhibition of neointimal formation.19,20 On the other hand, a new concept has evolved that a fundamental pathologic 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.41,42 In particular, programmed cell death/apoptosis is not a rare phenomenon, and it also occurs in pathophysiologic conditions such as after vascular injury by either mechanical or biochemical means.43 Therefore, it is becoming increasingly apparent that many factors may influence apoptosis in vascular cells. Among the many genes that have been suggested to be required by the molecular mechanism dictating apoptotic death, some have been shown to function as pacemakers to pave the way for cells either to live or to die. Probably, expressions of immediate early genes associated with the G1 phase of the cell cycle are candidates for this function. As reported previously, a well-known key regulator for halting the cell cycle at the G1/S interphase, p21, is involved in the execution of suicidal cell death in quiescent mouse 3T3 fibroblasts.23 However, postulated roles for p21 in apoptosis remain controversial, because both induction and protection from apoptosis were reported previously.23–25 Moreover, none of the reports mentioned the induction of apoptosis by p21 in VSMC. We therefore tested the hypothesis that overexpression of p21 gene would result in the inhibition of VSMC through the induction of apoptosis. Inhibition of human aortic VSMC by p21 overexpression was accompanied by the induction of apoptosis in human VSMC. This is the first study to demonstrate induction of apoptosis mediated by p21 in human VSMC. The number of apoptotic cells was relatively low, although transfection efficiency of HVJ-liposome method into VSMC is approximately 80–90% (unpublished observation; 31). Overexpression of p21 gene may not induce apoptosis in all cells, which is different from the inhibition of cell cycle progression. Probably, cells may undergo apoptosis only in certain conditions, such as inappropriate cell cycle progression. To elucidate the molecular mechanisms of induction of apoptosis by p21, we measured bcl-2 and bax proteins. 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.44,45 The bcl-2 and bax proteins can form heterodimers in cells.40 Our data revealed the inappropriate induction of bax to bcl-2, because overexpression of p21 resulted in a significant increase in bax protein and no change in bcl-2 protein. In the present study, we showed that upregulation of p21 is functionally related to the operational efficiency of the apoptotic process in human aortic VSMC transfected with p21 vector to increase the endogenous p21 protein level, although the susceptibility to apoptosis may be different among species of VSMC (p53-sensitive human and rabbit VSMC, and p53-resistant rat VSMC).46 An additional increase of p21 beyond the base level, seen in serum-stimulated human VSMC, may be involved in the molecular events precipitating a rapid program of cell demise. Although there is no apparent evidence of induction of apoptosis by overexpression of p21 in vivo, the present results suggest that induction of apoptosis through bax pathway may at least participate in the inhibition of VSMC in vitro. Apoptosis occurs within the context of restenosis after angioplasty in human subjects and experimental animals.41–43 Yang et al reported induction of endogenous p21 in balloon-injured porcine arteries;20 therefore, induction of p21 in vessels may contribute to cell death by apoptosis.
On the other hand, the induction of p21 has been reported to be coupled to the expression of early differentiation markers in myoblasts.24 Induction of cdk inhibitors may serve to maintain differentiated myocytes. It is recognized that VSMC involved in neointimal hyperplasia undergo a phenotypic change from a contractile to a secretory state, and these "activated" neointimal VSMC may release numerous cytokines that could, in turn, influence endothelial function and stimulate atherogenesis. Therefore, redifferentiation from neonatal to adult VSMC may have therapeutic value in inhibition of neointimal formation. As reported previously,21 overexpression of p21 gene in a vein graft model induced expression of the adult phenotype of VSMC in the formed neointima, which is less "synthetic" and closer to normal vascular structure. In addition to the inhibition of cell growth accompanied by apoptosis, the reexpressed adult-type phenotype might contribute to the inhibition of neointimal hyperplasia. Overall, these results demonstrated that inhibition of VSMC growth by overexpression of human p21 gene was accompanied by induction of apoptosis through an inappropriate increase in bax protein, which suggests that regulation of the cell cycle may be closely linked to apoptosis in human VSMC.
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Acknowledgments |
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We wish to thank Chihiro Noguchi for excellent technical assistance. Dr. Ryuichi Morishita is the recipient of a Harry Goldblatt Award from the Council of High Blood Pressure, American Heart Association. This work was partially supported by grants from the Ministry of Education of Japan, the Japan Society for the Promotion of Science, the Osaka Kidney Foundation (OKF 96-0002), ONO Medical Research Foundation and Japan Heart Foundation-Pfizer Pharmaceuticals Grant for Research on coronary artery disease.
Received September 16, 1997; first decision October 24, 1997; accepted October 31, 1997.
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