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Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:973-981

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:973.)
© 2000 American Heart Association, Inc.

Vascular Biology

p53, p21WAF1/CIP1, and MDM2 Involvement in the Proliferation and Apoptosis in an In Vitro Model of Conditionally Immortalized Human Vascular Smooth Muscle Cells

J.-Kuang Hsieh1; Dimitris Kletsas1; Gerard Clunn; Alun D. Hughes; Michael Schachter; Catherine Demoliou-Mason1

From the Department of Clinical Pharmacology (G.C., A.D.H., M.S., C.D.-M.), National Heart and Lung Institute, Imperial College of Science, Technology, and Medicine, St. Mary’s Hospital, London, UK; the Ludwig Institute (J.-K.H.), St. Mary’s Hospital, London, UK; and the Institute of Biology (D.K.), NCSR "Demokritos," Athens, Greece.

Correspondence to Dr A.D. Hughes, Clinical Pharmacology, National Heart and Lung Institute, Imperial College of Science, Technology, and Medicine, St. Mary’s Hospital, London W2 1NY, UK. E-mail a.hughes{at}ic.ac.uk


*    Abstract
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Abstract—Using an in vitro model of a conditionally immortalized cell line, we have investigated how human vascular smooth muscle cells (VSMCs) are affected by the expression of simian virus 40 (SV40) large T antigen (LT antigen), which binds to cell cycle regulators such as the tumor suppressor protein p53. Cells were obtained after infection of saphenous vein-derived VSMCs with a nonreplicative retroviral vector containing a temperature-sensitive mutant of SV40 LT antigen and were shown to have maintained some characteristics and responses of VSMCs. Under permissive-temperature conditions (36°C), the increased rate of cell proliferation was shown to be associated with expression of LT antigen and with LT antigen binding to and inactivation of p53. p53 inactivation failed to block apoptosis induced by serum withdrawal or UV irradiation. Downregulation of LT antigen expression at a nonpermissive temperature (39°C) was shown to be associated with growth arrest, increased expression of the cell cycle inhibitor p21WAF1/CIP1, increased murine double minute-2 promoter activity, and differential expression of murine double minute-2 gene products, suggesting that p53-induced transcription/transactivation may be involved in VSMC cycle control but not necessarily in apoptosis. The established SMC line HVTs-SM1 may be a useful model for study of the processes involved in myointimal hyperplasia and cellular aging, as well as for the study of cell cycle control in general.


Key Words: vascular smooth muscle • SV-40 • p53 • MDM2 • p21WAF1/CIP1


*    Introduction
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 *Introduction
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Vascular smooth muscle cell (VSMC) proliferation has long been considered to be a key event in remodeling of the vascular wall. Remodelling occurs during development, with vascular injury in diseases like atherosclerosis, and in vascular restenosis after invasive intervention.1 2 More recently, the precise role of cell proliferation in atherogenesis has received more critical attention,2 and other processes such as contraction, cell migration, and apoptosis have also been proposed to be involved. The mechanisms regulating cell cycle control in VSMCs remain largely unknown.2 Primary VSMCs especially those of human origin, are difficult to study in vitro: their availability is limited, they grow slowly, and they have a limited life span. Furthermore, cell responses may vary between cell lines depending on the donor, disease, or tissue.2 3 Recently, there has been an interest in in vitro models of established VSMC lines obtained from animal or human primary cells by transfection with simian virus 40 (SV40) large T (LT)-antigen or other viral oncogenes.4 5 6 7 However, although there are some reports of immortalized cell lines derived from animal cells, human cells in general have been more difficult to immortalize.

We describe herein some of the cellular characteristics of an SMC line derived from human saphenous vein, which was established by infection with a temperature-sensitive mutant SV40 LT antigen. Rapid proliferation of cells was associated with LT antigen expression and LT antigen–dependent inactivation of G1/S checkpoint control via inactivation of cell cycle regulators like p53.8 Downregulation of LT antigen expression under nonpermissive temperature conditions was shown to be associated with growth arrest, the appearance of morphological characteristics typical of senescent cells, and upregulation of the expression of growth regulator(s) downstream of p53, such as p21WAF1/CIP1.9 Transformed cells showed high levels of apoptosis, which increased after serum withdrawal or DNA damage by UV irradiation. Because this cell line maintains some characteristics of human VSMCs it may provide a useful in vitro model to study the regulation of phenotype and to investigate changes in cell cycle control involved in cellular aging, cell death, and other processes contributing to vascular disease.


*    Methods
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 *Methods
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Reagents, Antibodies, and Plasmids
All culture media were from Gibco Life Technologies/BRL. [methyl-3H]Thymidine (740 GBq/mmol) was from ICN Flow. Unfractionated porcine heparin (Paynes & Byrne) was a gift from Dr B. Mulloy, National Institute of Biological Standards and Control, South Mimms, UK. Platelet-derived growth factor-BB, epidermal growth factor, and transforming growth factor-ß were from Life Technologies. All other reagents used, unless otherwise specified, were from Sigma Chemical Co. Primary and secondary antibodies were from DAKO A/S. The p53 (non–cross-reactive) human-specific monoclonal antibody (mAb) DO-1,10 the p21WAF1/CIP1-specific mAb pAb53G817,11 the murine double minute-2 (MDM2)-luciferase (luc) reporter plasmid, and the independent reporter plasmid cytomegalovirus (CMV)–ß-galactosidase were kindly donated by Dr X. Lu (Ludwig Institute for Cancer Research, London, UK) and the SV40 LT antigen–specific mAbs 419 and 42312 by Dr T. Kamalati (Haddow Laboratories, Institute for Cancer Research, Sutton, UK). The mAb SM-E713 for smooth muscle myosin heavy-chains type 1 and 2 was a gift from Prof P. Pauletto (Clinica Medica I, Padova, Italy).

Cell Culture Conditions: Transfection and Isolation of Conditionally Immortalized Human VSMCs
Human SMCs were isolated from explants derived from normal saphenous vein from patients undergoing coronary artery bypass surgery as previously described.3 Cells that migrated from these explants were subcultured at a split ratio of 1:2 in Dulbecco’s modified Eagle’s medium (DMEM) containing 15% fetal calf serum (FCS). Equal numbers of cells from 12 separate lines derived from human saphenous vein–derived SMCs with similar proliferative responses were pooled at passage 2, subcultured, used at passage 3 or 4, and are referred to in this study as normal or untransformed VSMCs.

Actively growing primary VSMCs obtained from a single middle-aged patient were infected at passage 4 with conditioned medium from the amphotropic packaging PA317 cell line by using polybrene (8 mg/mL). The PA317 cell line produces the replication-defective helper virus–free vector pZipSV40-U19-5, which has been constructed by inserting at the single BamHI restriction site of the vector pZipNeoSV(X) a full-length SV40 LT antigen carrying both the temperature-sensitive A58 and U19 mutations.14 The temperature-sensitive A58 mutation confers temperature sensitivity for LT antigen expression, and the U19 mutation disables replication of the retroviral vector.14 15 Seven days after infection, the cells were put under G418 (500 µg/mL) selection. The infection was carried out by Dr M. J. O’Hare at Haddow Laboratories, Institute of Cancer Research, Sutton, Surrey, UK. Transformed cells that survived G418 selection were subcultured in DMEM containing 10% FCS and 200 µg/mL G418. Infection, selection, and maintenance of cells were carried out at the permissive temperature of 36°C. After {approx}100 population doublings, the cells were subcloned by serial dilution because there was no apparent crisis. The various clones obtained had a similar morphology, but only 20% survived subculturing for an additional 10 passages. One clone, designated herein as HVTs-SM1, has been in continuous culture for >2 years, exceeding 200 population doublings.

Plasmid Transfection and luc Assay
HVTs-SM1 cells plated in 3-cm-diameter dishes (104/dish) in 10% FCS–DMEM were transfected 24 hours later with 1 to 2 µg of MDM2-luc reporter plasmid by using the calcium phosphate precipitation method.9 An equivalent amount of CMV–ß-galactosidase plasmid was cotransfected for normalization of transfection efficiency. The transfected cells were incubated at 36°C or 39°C, and at the indicated time points they were washed 3 times with PBS, lysed in 100 µL of lysis buffer (Promega UK Ltd), and assayed for reporter activity of luc and ß-galactosidase.16

DNA Synthesis and Cell Proliferation
Normal and HVTs-SM1 cells were plated at 36°C in 96-well plates at 104 cells/well in NCTC-109 medium containing 10% FCS (10% FCS–NCTC-109). Twenty-four hours later, the cells were washed 3 times with PBS and growth-arrested (72 hours) in serum-free medium. DNA synthesis induced by the various stimulants was measured 30 hours after stimulation by using 5 µCi [methyl-3H]thymidine that was added 6 hours before harvesting the DNA, as previously described.17 Cell growth rates were measured by plating 5x104 cells/cm2 in 6-well plates in 10% FCS–NCTC-109 medium. Twenty-four hours later, the cells were growth-arrested in serum-free NCTC-109 medium for 72 hours. Then the medium was replaced with serum-free medium or 10% FCS–containing medium, in the presence or absence of 150 µg/mL heparin, and the cells were incubated at 36°C or 39°C with medium changes every 3 days. At the indicated time points, cells were trypsinized and counted with a Coulter counter. In addition, in some studies, cells synthesizing DNA were also identified after dual labeling with bromodeoxyuridine and Hoechst 33258 dye. In brief, cells plated on glass coverslips at different densities in 24-well plates overnight were growth-arrested as above and labeled with 50 µmol/L bromodeoxyuridine added in fresh serum-free medium for the last 24 hours. The cells were fixed in freshly prepared 4% paraformaldehyde/PBS (10 minutes), permeabilized with 0.2% Triton X-100/PBS (5 minutes), treated with 2N HCl (10 minutes), incubated with fluorescein-conjugated anti-bromodeoxyuridine mAb/PBS (30 minutes), and then stained with 1 µg/mL Hoechst 33258/PBS (30 minutes) in the dark at room temperature. Cells were washed 3 times with PBS at each step. Hoechst 33258– and bromodeoxyuridine-positive nuclei were counted on a Zeiss fluorescence microscope with a 20x objective; a field containing {approx}200 cells was used for quantification purposes.

Immunofluorescence Microscopy
Cells were plated on glass coverslips and were grown in 10% FCS–DMEM. They were washed with PBS, fixed in ice-cold ethanol or a methanol/acetone (1:1, vol/vol) solution (10 minutes), washed 5 times with PBS, and air dried. Then they were incubated with primary antibody (diluted 1:50 or 1:100 in 10% serum) overnight at 4°C, washed 3 times for 10 minutes with PBS and incubated with FITC-conjugated anti-mouse or anti-rabbit IgG (1:50) in PBS at 37°C (1 hour). After being washed, the cells were stained with Hoechst 33258/PBS (1 ng/ml, 10 minutes) and observed under a fluorescence microscope.

Immunoprecipitation and Immunoblotting
Subconfluent cultures of normal or HVTs-SM1 cells grown at 36°C were growth-arrested in serum-free NCTC-109 medium, which was replaced after 7 hours with either fresh serum-free or 10% FCS medium. After incubation at 36°C or 39°C for the indicated periods, the cells were washed 3 times with ice-cold PBS and lysed with 100 µL/106 cells of lysis buffer. Proteins in cell lysates were resolved by electrophoresis on 10% or 12.5% SDS-polyacrylamide gels, followed by Western blotting on nitrocellulose membranes (Schleicher and Schuell). In brief, the membranes were blocked with 10% nonfat milk in 10 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, and 0.05% Tween-20 (TTBS) buffer; washed with TTBS, and incubated with the primary antibody at room temperature for 1 hour or at 4°C overnight. Then they were incubated (1 to 2 hours) with horseradish peroxidase–conjugated secondary antibody (1:1000) in TTBS at room temperature. Immunoreactive bands were visualized on Kodak X-OMAT AR film by chemiluminescence (ECL kit) according to the manufacturer’s (Amersham) instructions.

For immunoprecipitation, cell lysates (1.0 µg) were precleared with protein G–Sepharose beads (10 µL) and mixed with 5 to 10 µg of purified antibody or 1 to 2 mL of ascites at 4°C overnight. Antibody complexes were precipitated with 10 µL of protein G–Sepharose beads at 4°C (1 to 2 hours). After the beads were washed 3 times with lysis buffer and twice with the same buffer without NP-40, bound immunocomplexes were extracted by boiling (3 minutes) in 20 µL of 2x-concentrated SDS–polyacrylamide gel electrophoresis sample buffer, electrophoresed, and immunoblotted.

UV Irradiation and Cell Apoptosis
Normal and HVTs-SM1 cells were irradiated at 50 mJ/cm2 in an XL-1000 UV cross-linker (Spectronics Corp) and then incubated at 36°C in 10% FCS–DMEM. At the indicated time points they were trypsinized and counted. In parallel, the detached and adherent cells from each of 2 additional wells were collected for flow cytometry, washed with PBS, fixed in 70% ice-cold ethanol, and centrifuged at 2000 rpm (10 minutes 4°C). Cells and apoptotic bodies in each pellet were resuspended in PBS containing 50 µg/mL propidium iodide and 12 µg/mL RNase A and incubated in the dark (30 minutes, room temperature). Flow cytometry was performed in a FACSORT (Becton-Dickinson), and fluorescence signals were plotted by using Cell Quest software.

Statistics and Data Analysis
All data are presented as mean±SD of n observations. Statistical comparisons were made with a Student’s t test; a value of P<0.05 was considered significant.


*    Results
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 *Results
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Conditionally Immortalized Human Vein–Derived SMCs
Normal VSMCs (passaged at a 1:3 split ratio) stopped dividing after 10 to 15 passages and became senescent. The transformed HVTs-SM1 cells, however, have been in continuous culture at the permissive temperature for >200 population doublings. The transformed cells had lost the elongated, spindlelike shape of normal VSMCs and were smaller with a morphology ranging from polygonal epithelioid-like to fibroblast-like (Figure 1ADown versus 1B). When HVTs-SM1 cells were transferred to 39°C, after {approx}5 to 7 days, they acquired morphological characteristics more similar to those of normal cells (Figure 1CDown), except that they were enlarged and flattened, a characteristic of in vitro senescent cells.18 19



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  		Figure 1. Comparison of cell morphologies of normal VSMCs and transformed cells (HVTs-SM1). Phase-contrast microscopy of (A) normal confluent and (B) transformed confluent cells cultured at 36°C and (C) transformed confluent cells cultured at 39°C. Magnification x100.

We investigated whether the transformed HVTs-SM1 cells retained cytoskeletal proteins typical of SMCs. The majority of transformed cells stained positively for smooth muscle {alpha}-actin. The decorated filaments appeared to be more cytoplasmic and irregular in the transformed cells, whereas in normal cells they traversed the cell along its long axis (Figure 2Down: A1, A2) There were no observable differences in the strength of smooth muscle {alpha}-actin staining and filament distribution when either normal or transformed VSMCs were incubated at 39°C for 24 to 48 hours. Staining for vimentin was positive in both cell types, but compared with normal cells, the filaments of the transformed cells were more sparse, less organized, and appeared spread from the perinuclear bundles to the periphery of the cell (Figure 2Down: B1, B2). Antibody staining for von Willebrand factor and desmin was negative for both normal and transformed cells. The transformed cells showed weak and mainly perinuclear rodlike staining for smooth muscle myosin heavy chain (Figure 2Down, C2), whereas in normal VSMCs, the staining was observed in organized filaments along the long axis of the cell (Figure 2Down, C1).



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  		Figure 2. Immunofluorescence staining of smooth muscle cytoskeletal proteins. Normal SMCs (column 1) and transformed HVTs-SM1 cells (column 2) cultured at 36°C were assessed for smooth muscle–specific {alpha}-actin (A), vimentin (B), and smooth muscle myosin heavy chain (C). Magnification x400.

Expression of LT Antigen and Cell Cycle Regulatory Molecules in HVTs-SM1 Cells
Previous reports on cells immortalized with SV40 have identified that the expression of a functional LT antigen is required for maintenance of the immortalized phenotype.15 Those studies also established that SV40 LT antigen binds to wild-type p53 and extends its half-life.8 20 We therefore investigated whether HVTs-SM1 cells expressed LT antigen conditionally and whether LT antigen expression had an effect on p53 protein levels. Indirect immunofluorescence with anti–LT antigen or with anti–human-specific wild-type p53 antibodies in cells grown at 36°C showed positive nuclear staining for both antigens in all cells (Figure 3Down). LT antigen and p53 nuclear immunofluorescence staining were reduced to almost background levels when cells were transferred to 39°C (Figure 3Down: A2, B2), and normal cells grown under similar conditions showed no nuclear staining with either antibody. Similarly, immunoblotting studies showed elevated levels of LT antigen and p53 in cells grown at 36°C but not in cells cultured at 39°C (Figure 4Down). Levels of LT antigen and p53 were unaffected by the presence or absence of 10% FCS, and p53 was found to coimmunoprecipitate with LT antigen (Figure 4Down). It should be noted that the HVTs-SM1 cells had to be cultured for 5 to 7 days at 39°C before the protein levels of LT antigen and p53 were reduced below the detection limits of the antibodies used.



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  		Figure 3. LT antigen and p53 immunofluorescence staining. Transformed HVTs-SM1 cells cultured at 36°C (A1, B1) or 39°C (A2, B2) were stained with LT antigen mAb (A) or with p53 mAb (B). Magnification x400.



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  		Figure 4. Immunoblot analyses of LT antigen, p53, and p21 protein expression in HVTs-SM1 cells. For LT antigen, Western blots of lysates from cells grown for 9 days in the presence (lanes 1, 2) or absence (lanes 3, 4) of 10% FCS at 36°C (lanes 1, 3) or 39°C (lanes 2, 4). For p53, Western blots of lysates from cells grown for 9 days in the presence (lanes 1, 2) or absence (lanes 3, 4) of 10% FCS at 36°C (lanes 1, 3) or 39°C (lanes 2, 4). For p53/LT antigen for detection of LT antigen–p53 complex, lysates from cells cultured for 5 days at 36°C (lanes 1, 3) or 39°C (lanes 2, 4) were first treated with anti-p21 mAb (lanes 1, 2; control) or anti–LT antigen mAb (lanes 3, 4), and the immunoprecipitates were then subjected to Western blotting by using an anti-p53 mAb. For p21, Western blots of HVTs-SM1 cells grown for 7 days at 36°C (lane 1) or 39°C (lane 2), transformed human aortic SMCs that do not express p21 (negative control) cultured at 36°C and 39°C (lanes 3, 4, respectively); positive controls, human lymphoma cells (lane 5); mouse fibroblast cells (lane 6).

We also examined the transcription of molecules that are known to be regulated by p53, such as p21 and MDM2, because it had been documented that LT antigen binding to p53 may adversely affect its function.20 The specific mAb to the cell cycle inhibitor p21 detected a 21-kDa protein band when the transformed cells were cultured at 39°C only (Figure 4Up). Taking into account the observed temperature-dependent LT antigen and p53 protein expression profiles and the coimmunoprecipitation of LT antigen with p53, these results are in keeping with the proposal that p53-induced p21 expression may be inhibited by LT antigen binding to p53.9

To investigate the effect of LT antigen expression on the transcriptional regulation of MDM2 by p53, HVTs-SM1 cells were transfected with an MDM2-luc reporter plasmid. The luc activity profile obtained showed a 4-fold increase in MDM2-promoter activity when the cells were cultured at 39°C rather than at 36°C (Figure 5Down). Furthermore, Western blot analysis of HVTs-SM1 lysates from cells cultured at the 2 temperatures indicated that MDM2 protein levels were also reduced in HVTs-SM1 cells cultured at permissive temperature (Figure 5Down, insert).



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  		Figure 5. Expression of MDM2 promoter activity in HVTs-SM1 cells. HVTs-SM1 cells (104) were plated in 3-cm dishes in 10% FCS in triplicate, transfected 24 hours later with MDM2-luc reporter plasmid, and subsequently incubated at 36°C or 39°C. The cells were harvested 12 or 24 hours later, lysed, and assayed for luc activity. Results are the mean±SD of 1 of 2 representative experiments. Insert, Anti-MDM2 mAb Western blot of lysates from transformed cells that were cultured for 5 days at 36°C (lane 1) or 39°C (lane 2).

Growth and Survival of HVTs-SM1 Cells
We then investigated the growth rate and survival of HVTs-SM1 cells. At 36°C, the serum–growth response curves of transformed (Figure 6ADown) and normal (Figure 6DDown) cells plated at 5x104 cells/cm2 appeared sigmoidal. The transformed cells, however, grew more rapidly (doubling time of 49±9 hours, n=4; P<0.001) and reached a density that was {approx}6-fold higher than that of normal cells (doubling time of 132±36 hours, n=12, passage 3). Under permissive-temperature conditions, the serum-dependent proliferation of HVTs-SM1 cells, like that of normal cells, appeared to decrease through contact inhibition (Figure 6ADown versus Figure 6DDown). Anti-bromodeoxyuridine antibody labeling and acridine orange staining showed that cell division as well as cell death by apoptosis was taking place in HVTs-SM1 cells concurrently during this period (data not shown). After a long incubation period (3 to 4 months), normal VSMCs continued to proliferate very slowly and form a densely packed multilayer, whereas the transformed cells did not form such a multilayer.



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  		Figure 6. Proliferation of HVTs-SM1 cells (A through C) and normal SMCs (D) under permissive and nonpermissive temperature conditions. Cells plated at 1x104/cm2 (squares), 2.5x104/cm2 (diamonds), or 5.0x104/cm2 (circles) were growth-arrested in serum-free medium at 36°C for 72 hours. Then they were cultured in 10% FCS (filled symbols) or serum-free medium (open symbols) at 36°C (A, C, D) or 39°C (B). Media were replaced every 3 days. Results are the mean±SD of triplicate or quadruplicate determinations of 1 representative experiment repeated 2 or 3 times. E and F, Apoptosis in HVTs-SM1 cells at 36°C as visualized by fluorescence microscopy of cells stained with 15 µg/mL acridine orange 96 hours (E) and 144 hours (F) after serum withdrawal. Magnificationx400.

Unlike the situation with nontransformed cells, serum withdrawal under permissive conditions did not cause the HVTs-SM1 cells to growth-arrest (>90% of cells were bromodeoxyuridine-positive 24 hours later). However, the cells were unable to sustain continuous proliferation, their numbers declined (Figure 6CUp), and acridine orange staining demonstrated many apoptotic cells during this period (Figure 6EUp and 6FUp). In contrast, serum withdrawal from normal SMCs induced only a small loss of cells within the first 24 to 48 hours. The remaining cells appeared to survive in a growth-arrested state for at least 1 week (Figure 6DUp).

The temperature change to 39°C appeared to have no effect on the serum-dependent proliferation of HVTs-SM1 cells in the first few days. However, cell number declined 5 to 7 days later (Figure 6BUp), and acridine orange staining indicated increased apoptosis. The fate of surviving cells depended on their density. If the cells did not become very sparse after the period of rapid cell death, they continued to proliferate very slowly and remained viable for at least 3 to 4 months. Flow cytometry indicated that the majority of cells accumulated at the G1 as well as G2 phases of the cell cycle (the TableDown), in agreement with previous reports.15 With time, there was a significant increase in cell size and the appearance of perinuclear vacuoles, and these cells resembled in vitro senescent or atherosclerotic cells.18 19 Provided that the temperature was lowered to 36°C before the cells became very sparse, they were able to resume very quickly as vigorous a growth in response to serum as that of cells that had not been exposed to the nonpermissive temperature.


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  		Table 1. Flow-Cytometric Analyses of UV-Irradiated Normal VSMCs and HVTs-SM1 Cells

We also investigated whether HVTs-SM1 cells retained responses to smooth muscle growth factors and heparin. As shown in Figure 7ADown, under permissive-temperature conditions, induction of HVTs-SM1 cells by 10% FCS had a large positive effect on thymidine incorporation, whereas induction with platelet-derived growth factor and epidermal growth factor had only a small positive effect; transforming growth factor-ß had no apparent effect. Heparin (150 µg/mL) inhibited thymidine incorporation induced by serum by {approx}50% and that induced by platelet-derived growth factor and epidermal growth factor to a lesser extent (Figure 7ADown). Heparin inhibition of serum-induced DNA synthesis was broadly comparable to what we previously observed in normal VSMCs,3 and no further effect was seen after an increase in heparin concentration up to 5-fold. Serum-induced cell proliferation was inhibited by 25% to 30% when cells were cultured in the presence of 150 µg/mL heparin for 7 days (Figure 7BDown). When cells were cultured in the presence of 0.5% serum, addition of heparin (150 µg/mL) resulted in a small net reduction in cell number (Figure 7BDown versus Figure 6CUp).



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  		Figure 7. DNA synthesis and proliferation of HVTs-SM1 cells induced by serum and growth factors in the presence and absence of heparin. A, Cells plated at 5x104 in 96-well plates at 36°C were growth-arrested for 48 to 72 hours in serum-free medium and then induced with either 10% FCS or 5 ng/mL growth factor in medium only, in the absence (unfilled bars) or presence (filled bars) of 150 µg/mL heparin. B, Time-dependent proliferation of cells plated at 5x104 in 35-mm-diameter plates. Cells were incubated with 10% FCS (circles) or 0.5% FCS (squares) in the absence (unfilled symbols) or presence (filled symbols) of heparin (150 µg/mL). Results represent the mean±SD of quadruplicate determinations of 1 of 2 representative experiments.

LT Antigen Transformation Increases SMC Sensitivity to UV Irradiation
Flow cytometry of UV-irradiated (50 mJ/cm2) normal SMCs cultured at 36°C, showed no significant time-dependent increase in the percentage of apoptotic cell bodies (control, 0 time, 10%; 24 hours after irradiation, 7%), suggesting that the cells were resistant to UV irradiation. The majority of cells remained in the G1 phase of the cell cycle, with only a small proportion of cells transversing the S phase (the TableUp), in keeping with the slow proliferation rate of normal SMCs. In contrast, the transformed cells showed a 6-fold increase in the percentage of apoptotic bodies as early as 6 hours after irradiation (control, 0 time, 6%; 6 hours after irradiation, 34%). This was accompanied by a time-dependent increase in degraded DNA and a decrease in the percentage of cells in the G1 phase. UV irradiation had no significant effect on the proportion of cells traversing the S phase. However, 24 hours after irradiation, there was an increase in the proportion of cells in the G2 phase (the TableUp). Compared with nonirradiated HVTs-SM1 cells, there was an {approx}40% to 50% reduction in cell numbers 48 to 50 hours after irradiation; this was accompanied by a period of growth arrest and then recovery (Figure 8Down). Early apoptosis of UV-irradiated HVTs-SM1 cells was also confirmed by detection of DNA fragmentation 4 hours after irradiation (data not shown).



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  		Figure 8. Proliferation of UV-irradiated HVTs-SM1 cells. UV-irradiated (open symbols) and nonirradiated (filled symbols) cells cultured in 10% FCS/NCTC at 36°C for the indicated time points were harvested and counted. Results are the mean±SD of triplicate determinations of 1 of 2 experiments.


*    Discussion
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 *Discussion
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In vitro aging and senescence limit the life span of primary human VSMCs.18 19 This limitation and the interindividual and intraindividual variabilities of cellular responses to growth factors/inhibitors3 emphasize the need for established cell lines. However, in contrast to rodent cells, only a few such human cell lines have been established by viral infection.21 In a previous study that used SV40 infection of human VSMCs, the maximum life span reported was {approx}60 population doublings.22 Using a temperature-sensitive replication-defective SV40 vector, we have been able to establish an immortal (life span >200 population doublings) human VSMC line that we have named HVTs-SM1. These cells are conditionally immortalized because their proliferative phenotype is expressed only under the permissive temperature. Under nonpermissive-temperature conditions, the cells growth-arrest and eventually lose viability in a density-dependent manner, implying the presence of an autoregulatory mechanism for survival. The transformed cells are smaller and have lost the typical spindle morphology that characterizes normal VSMCs. Nevertheless, the cells continue to express smooth muscle–specific cytoskeletal proteins, including myosin heavy chain and {alpha}-actin. We have also shown at permissive temperatures that HVTs-SM1 cells retain, to some extent, their response to SMC growth factors, like platelet-derived growth factor-BB, and to growth inhibitors like heparin.3 23 However, DNA synthesis in response to platelet-derived growth factor-BB was 2- to 3-fold lower than in normal cells, possibly as a result of the LT antigen–induced downregulation of platelet-derived growth factor receptor expression.24

SMCs play an important role in the pathogenesis of atherosclerosis, vascular graft occlusion, and restenosis after angioplasty. Although the precise role of smooth muscle proliferation and apoptosis in the development of these diseases, particularly atherosclerosis, is debated,2 25 it is likely that these processes contribute, at least at some stage, to their pathogenesis. HVTs-SM1 cells may be a useful model to study the role of growth regulators in normal and pathological VSMC proliferation, cell death, and other aspects of cell cycle control.

SV40 LT antigen is known to have transforming and tumorigenic potential, because it is able to target and inactivate several molecules involved in the control of cell cycle progression, including the tumor suppressor molecules Rb and p53.20 p53 mediates transcription of the cyclin-dependent kinase inhibitor p21WAF1, and this is a key event in cell growth arrest and cell apoptosis due to DNA damage.9 p21 regulates the activity of Rb by inhibiting its phosphorylation, the subsequent release of the E2F family members, and finally the cell’s entry into the S phase.26 p53 is also known to interact with the tumor progression factor MDM2,27 28 which promotes p53 degradation and can inhibit p53 transactivation and transrepression.29 30 Interestingly, both p53 and MDM2 have been reported to be expressed in SMCs and macrophages in human atherosclerotic tissue,31 and high levels of p53 in association with CMV infection were seen in 1 study of coronary restenosis.32 Our results indicate that the rapid and continuous proliferation of HVTs-SM1 cells under permissive-temperature conditions is associated with the expression of SV40 LT antigen and with LT antigen binding and inactivation of p53. This conclusion was based on the evidence that the expression of p21WAF1 protein and of MDM2 promoter activity was inhibited under these conditions. Because Rb activity is regulated by p21-dependent phosphorylation, deregulation of p21 expression would also be expected to affect Rb activity. In addition, as previously reported, direct binding of LT antigen to Rb may release E2F transcription factor(s) and thus, contribute further to the deregulation of cell growth arrest and apoptosis.33 Deregulation of cell cycle control at the level of p53 and of growth regulators that depend on p53 activity in HVTs-SM1 cells is further suggested by the fact that when LT antigen is no longer functional owing to a temperature shift to 39°C, the cells were able to growth-arrest. Under these conditions, the cells express high levels of p21, as is observed in senescent cells in vitro.34 Furthermore, cells remained viable, could be rescued, and resumed proliferation even several weeks later after return to the permissive temperature.

Apoptosis of VSMCs has been observed in vasculature remodeling during development and may be another important mechanism in the modulation of cellularity in vascular disease.35 We observed that proliferation of HVTs-SM1 cells at the permissive temperature was accompanied by apoptosis. Compared with normal cells, apoptosis of transformed cells was increased by serum withdrawal, suggesting that the cells are more dependent on serum factors for survival than for proliferation. It has been proposed recently that p53 is a potent inducer of apoptosis when Rb is inhibited in smooth muscle.22 However, the data from our system in which both Rb and p53 are inactivated by SV40 LT antigen (at the permissive temperature) imply the presence of a p53-independent pathway for apoptosis. This inference is supported by the conclusions of a recent study, which examined the effect of p53 inactivation on atherogenesis in apoE-knockout mice, an animal model for atherosclerosis.36 However, the cell death observed after cell transfer to the nonpermissive temperature may be attributable to the slow loss of SV40 LT antigen expression. UV irradiation induced rapid apoptosis in HVTs-SM1 cells grown at the permissive temperature, whereas normal cells did not undergo detectable apoptosis after UV irradiation. The reason for this difference is unclear but may be a result of their slow proliferation rate, which allows DNA repair to take place. This question requires further study, and examination of the effects of more pathophysiologically relevant mediators such as oxidized lipoproteins, oxysterols, and nitric oxide may be of particular interest. Apoptosis after DNA damage by UV irradiation has also been associated with increased p53 activity,37 but from the results of the present study, it appears that p53-induced transcriptional activity is not required for UV-induced apoptosis. Overall, 1 possible hypothesis is that in HVTs-SM1 cells, E2F1 overexpression (due to inactivation of Rb by LT antigen) can induce apoptosis independently of p53, as previously shown in Saos-2 cells,27 which are null for p53 and lack a functional Rb. Further studies will be required to confirm this hypothesis.

In conclusion, we have shown that the established HVTs-SM1 cell line can be used as an in vitro model to study specific perturbations of cell proliferation. This model of human VSMCs offers opportunities to establish a better understanding of the processes and molecules involved in the aging and senescence of the vasculature and in the control of the cell cycle in human VSMCs.


*    Acknowledgments
 
This work was supported by the Thrombosis Research Trust (UK), the British Heart Foundation (UK), the British Council, the UK Home Office, and the Greek-Britain Scientific and Technological Collaboration. D. Kletsas was supported with an FEBS fellowship. We are grateful to M. J. O’Hare for the transfection of cells and K. Gallagher for assistance with tissue culture. We also thank H. Pratsinis and members of the Clinical Pharmacology Department, Imperial College, for their helpful comments and assistance.


*    Footnotes
 
1 J.-K.H., D.K., and C.D.-M. all contributed equally to this work. Back

Received March 11, 1999; accepted October 13, 1999.


*    References
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