© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
eNOS gene transfer to vascular smooth muscle cells inhibits cell proliferation via upregulation of p27 and p21 and not apoptosis
aDepartment of Endocrinology, Mayo Clinic and Foundation, Rochester, MN 55905, USA
bDepartment of Anesthesiology, Mayo Clinic and Foundation, 200 First Street SW, Rochester, MN 55905, USA
* Corresponding author. Tel.: +1-507-255-6768; fax: +1-507-255-4828 obrien.timothy{at}mayo.edu
Received 4 February 2000; accepted 9 May 2000
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Abstract |
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Objective: Smooth muscle cell (SMC) proliferation is a critical component of vascular diseases such as atherosclerosis and restenosis. Nitric oxide (NO) donors and gene transfer of endothelial nitric oxide synthase (eNOS) have been shown to inhibit SMC proliferation. NO may cause this effect by delaying cell cycle progression and/or induction of apoptosis. The aim of the current study was to examine the mechanism of eNOS-mediated inhibition of SMC proliferation. In addition, the effect of eNOS expression in vascular SMCs on the expression of the cyclin dependent kinase inhibitors, p27 and p21 was examined. Methods: SMCs were transduced with an adenoviral vector encoding eNOS (AdeNOS) or β-galactosidase (AdβGal) at a multiplicity of infection of 100. Non-transduced cells served as additional controls. Transgene expression was sought by NADPH diaphorase staining, immunohistochemistry and Western Blotting. Functionality of the recombinant protein was assessed by measurement of cGMP. Cell cycle analysis was performed by flow cytometry and p27 and p21 expression were studied by western blot analysis. Apoptosis was sought by Annexin V staining and DNA laddering. Results: eNOS expression was detected in transduced SMCs. cGMP levels were increased in eNOS-transduced compared to control cells. Expression of eNOS in SMCs resulted in a delay in cell cycle progression and upregulation of p27 and p21. There was no increase in apoptosis detected in eNOS transduced cells after 24 or 72 h. Conclusion: eNOS gene transfer to vascular SMCs inhibits cell proliferation via upregulation of p27 and p21 resulting in a delay in cell cycle progression.
KEYWORDS Nitric oxide; Smooth muscle; Gene therapy; Cell culture/isolation; Restenosis
This article is referred to in the Editorial by C.M. Holt (pages 640–641) in this issue.
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1 Introduction |
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Nitric oxide (NO) is generated from L-arginine via a family of nitric oxide synthase (NOS) enzymes [1]. Endothelial NOS is constitutively expressed and NO derived from this isoform in the vessel wall has a number of protective effects including inhibition of platelet and monocyte adhesion [2,3] and smooth muscle cell (SMC) proliferation [4,5]. Reduced NO generation due to decreased eNOS activity has been proposed to result in increased SMC proliferation after vascular injury [6–9]. Transfer of the gene for eNOS to the vessel wall may reverse this defect and thus inhibit SMC proliferation. Indeed, NO donors and eNOS gene transfer have been shown to inhibit SMC proliferation in vitro [4,10]. In addition, in vivo gene transfer of eNOS has been shown to reduce intimal hyperplasia after vascular mechanical injury in rat models [11,12] and iNOS gene transfer has been shown to have a similar effect [13]. The mechanism of NO-mediated inhibition of SMC proliferation may involve delay of cell cycle progression or induction of apoptosis. While NO donors have been shown to block cell cycle progression [14], the effect of NO on apoptosis has varied depending on the cell line studied. NO has been shown to inhibit apoptosis in endothelial cells and cardiomyocytes [15,16]. In contrast, NO donors were shown to dose dependently induce apoptosis in vascular SMCs [17]. The aim of the current study was to define further the mechanism of inhibition of smooth muscle proliferation following eNOS gene transfer to vascular SMCs and to assess the relative roles of delayed cell cycle progression and apoptosis.
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2 Methods |
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2.1 Construction, propagation, and purification of adenoviral vectors
A recombinant adenovirus encoding the eNOS gene driven by the cytomegalovirus promoter was generated as previously described [10]. Bovine eNOS cDNA was cloned into the pACCMVpLpA vector, a kind gift of Robert Gerard, University of Texas Southwestern Medical Center, Dallas, TX, USA). The resulting plasmid was linearized and cotransfected with dl309 into 293 cells by calcium phosphate/DNA coprecipitation. Recombinant adenoviral vectors were generated by homologous recombination. Viral plaques were picked and propagated in 293 cells. Viral DNA was enriched by Hirt extraction and screened by restriction mapping and polymerase chain reaction for the presence of eNOS cDNA. Positive plaques underwent two further rounds of plaque purification in 293 cells. Virus was purified by double cesium chloride gradient ultracentrifugation and was dialyzed against 10 mmol/l Tris, 1.0 mmol/l MgCl2, 1.0 mmol/l HEPES, and 10% glycerol for 4 h at 4°C. Viral titer was determined by plaque assay. A recombinant replication defective adenoviral vector encoding the E. coli β-galactosidase gene (AdβGal) driven by the cytomegalovirus promoter was obtained from Dr. James Wilson (University of Pennsylvania, Philadelphia, PA, USA) and used as a control. It was propagated, isolated, and quantitated as described above. Viral stocks were stored at –80°C.
2.2 Cell culture
Porcine coronary artery smooth muscle cells (PCSMCs) were obtained from the coronary arteries of domestic crossbred pigs by enzymatic digestion with 1% collagenase. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The arteries were excised aseptically from the animal and placed in serum-free medium M199. After removal of periadventitial fat, the vessels were cut longitudinally, opened flat, and incubated in medium containing collagenase for 20 min. The endothelial and adventitial surfaces were scraped with a cell scraper. The digested vessels were cut into small rings and placed in 60-mm diameter dishes in medium M199 containing 10% heat-inactivated fetal calf serum (FCS), L-glutamine (2 mmol/l), penicillin (100 U/ml), streptomycin (100 µg/ml), Earle's salts, and NaHCO3 (2.2 g/l). The medium was changed every 3 days and when cells reached 
70% confluence, they were trypsinized and replated in 75-cm2 flasks. Identity of the cells was confirmed by the typical ‘hill-and-valley’ appearance at confluence and positive smooth muscle 
-actin staining. The cells were kept at 37°C in humidified 5%CO2–95%O2. PCSMCs were passaged by trypsinization, and cells between the third and seventh passages were used for the experiments.
2.3 Transduction with adenoviral vectors
PCSMCs were plated at a density of 4–5x10 5/cm2 in a 60x15 mm dish or 3x106/cm2 in a 100x20 mm dish and cultured overnight in medium M199 with 10% FCS. On the following day, the cells were transduced with either an adenoviral encoding eNOS (AdeNOS) or β-galactosidase (AdβGal) at multiplicity of infection (MOI) of 100 or were left non-transduced. The vector was diluted in PBS with 0.5% albumin and cells were exposed to the vector for 1 h. The vector was then aspirated, the cells washed with PBS and medium was replaced.
2.4 eNOS immunoreactivity
For immunohistochemical detection of eNOS, PCSMCs were plated in chamber slides. Forty-eight hours after transduction with vectors at MOI 100, the media was aspirated. After immersion fixation in acetone (4°C) and drying, the slide was incubated in 0.1% sodium azide–0.3% hydrogen peroxide and then incubated with 5% goat serum–PBS–Tween 20 to block nonspecific protein binding sites. An eNOS monoclonal antibody (1:50 dilution, Transduction Laboratory) was applied for 60 min at room temperature, followed by incubations with biotinylated rabbit anti-mouse F (ab') 2 secondary antibody (dilution 1:300) (Dako) and peroxidase-conjugated streptavidin (dilution 1:300) (Dako). After a 30-s immersion in 0.1 mol/l sodium acetate buffer (pH 5.2), eNOS immunoreactivity was visualized with 3-amino-9-ethylcarbazole and hematoxylin counterstaining.
2.5 NADPH-diaphorase staining
Forty-eight hours after transduction of cells with vectors at MOI 100, cells were washed with PBS and then fixed in 4% para-formaldehyde for 5 min. A solution containing 1 mmol/l βNADPH, 0.5 mmol/l NBT, and 50 mmol/l Tris (pH 8.0) was added and the cells incubated for 30 min at 37°C. The solution was replaced with PBS, and the cells were assessed for staining by phase-contrast microscopy.
2.6 Western blot analysis of transduced PCSMCs for eNOS
eNOS expression was sought by western blot analysis of transduced and control cells. Forty-eight hours after transduction with vectors at MOI 100, western blot analysis was performed on whole-cell lysates by incubating PCSMCs in lysis buffer (50 mM sodium pyrophosphate, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 100 µM Na3VO4, 10 mM HEPES (pH7.4), 0.1% Triton X-100, 0.5 mM PMSF and 10 µg/ml leupeptin). Cellular debris was sonicated on ice, then followed by centrifugation at 14 000 g for 30 min to remove the insoluble pellet, and protein concentration was determined by bicinchoninic acid (BCA) assay (Pierce). A 50-µg amount of protein was fractionated by SDS–PAGE in a 7.5% gel. The resolved proteins were transferred to a 0.2 µm nitrocellulose membrane on a semidry electrophoretic transfer cell (Bio-Rad) for western blot analysis. Blots were blocked and incubated with a mouse anti-human eNOS monoclonal antibody (dilution 1:1500) (Transduction Laboratories) overnight at 4°C. After washing, secondary antibody (anti-mouse IgG, horseradish peroxidase-linked whole antibody (from sheep, dilution 1:1000) (Amersham Life Science)) was visualized using the ECL Western blotting detection system (Amersham Life Science).
2.7 Measurement of cGMP
PCSMCs were plated in 60x15 mm dishes and transduced at MOI 100 with Adβgal, AdeNOS or diluent alone. cGMP levels were measured 72 h later. Cells were washed with PBS twice and incubated at 37°C in 0.1 mmol/l isobutylmethylxanthine (IBMX) to inhibit degradation of the cyclic nucleotides by phosphodiesterases. cGMP was extracted under acidic conditions by adding 0.1 M HCl for 30 min and measured by radioimmunoassay (Amersham). To assess the effect on cGMP production under stimulated condition, calcium ionophore (1 µmol/l) and L-arginine (0.1 mmol/l) was added to the IBMX for 5 min prior to cGMP extraction. cGMP levels were normalized to the protein content of each dish, which was estimated by the BCA assay (Pierce).
2.8 Flow cytometry for cell cycle analysis
PCSMCs were plated in 60x15 mm dishes and cell cycle analysis was assessed by flow cytometry. Cells were transduced with AdeNOS, Adβgal or exposed to diluent alone. After 48 h of quiescence, cells were cultured in medium M199 with 10% FCS or left in medium with 0.5% FCS. Cells were incubated for an additional 24 h at 37°C. Cells were washed twice with PBS, and incubated for 1 h with permeabilization buffer containing Dnase-free Rnase and propidium iodide. Flow cytometry was performed using a FACScan model (Becton-Dickinson). The experiment was repeated on three separate occasions and the data analyzed by ANOVA using Fisher's test.
2.9 Western blot analysis for p27
p27 was sought by western blot analysis of transduced and control cells. PCSMCs were placed in 100x20 mm dish and transduced at MOI 100. Cells were cultured in medium M199 with 0.5% FCS for 48 h. Cells were then incubated for an additional 24 h in M199 medium with 0.5% or 10% FCS. Western blot analysis was performed on whole-cell lysates by incubating PCSMCs in 1 ml of lysis buffer (50 mM sodium pyrophosphate, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 100 µM Na3VO4, 10 mM HEPES (pH 7.4), 0.1% Triton X-100, 0.5 mM PMSF and 10 µg/ml leupeptin). Cellular debris was sonicated on ice, then followed by centrifugation at 14 000 g for 30 min to remove the insoluble pellet, and protein concentration was determined by BCA assay (Pierce). A 50-µg amount of protein was loaded on 12.5% SDS–PAGE. The resolved proteins were transferred to a 0.2-µm nitrocellulose membrane on a semidry electrophoretic transfer cell (Bio-Rad) for western blot analysis. Blots were blocked and incubated with a mouse anti-human Kip1/p27 monoclonal IgG1(dilution 1:2500) (Transduction Laboratories) overnight at 4°C and after washing were incubated with secondary antibody [anti-mouse IgG, horseradish peroxidase-linked whole antibody (from sheep) (dilution 1:5000, Amersham Life Science)]. The secondary antibody was visualized using the ECL Western blotting detection system (Amersham Life Science). Equal protein loading was confirmed by Ponceau Red staining.
2.10 Immunoprecipitation and western blot analysis for p21
p21 was detected by immunoprecipitation and western blot analysis of transduced and control cells. PCSMCs were plated in 100x20 mm dishes and transduced at MOI of 100, 24 h later. After 48 h medium was changed to medium M199 containing 0.5 or 10% FCS. After 24 h, whole-cell lysates were made by incubating each plate in 1 ml of lysis buffer (10 mM Tris–HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 50 mM NaF, 0.2 mM Na3VO4, 20 µg/ml leupeptin, 20 µg/ml phenylmethylsulfonyl fluoride and 0.5% (v/v) Igepol CA643). The lysates were sonicated on ice and centrifuged at 14 000 g for 30 min to remove insoluble material. All immunoprecipitation steps were carried out at 4°C. The supernatant was incubated with 4 µg mouse IgG 1 (Research Diagnostics) for 3 h, after which 25 µl Protein G-conjugated Sepharose (Pharmacia Biotec) was added for 2 h. After centrifugation to pellet the beads, the supernatant was incubated with 4 µg of mouse anti-p21 monoclonal antibody (Pharmingen) overnight. Immune complexes were captured with Protein G Sepharose and washed four times in lysis buffer. Anti-p21 immunoprecipitates were run on 12.5% SDS–PAGE, and protein transferred to 0.2-µm nitrocellulose membranes on semidry electrophoretic transfer cells (Bio-Rad). Blots were blocked overnight and incubated with the same anti-p21 monoclonal antibody overnight at 4°C. After washing, blots were incubated with horseradish peroxidase-linked anti-mouse IgG (Amersham Life Science) and processed for ECL (Amersham Life Science). Equal protein loading was confirmed by Ponceau red staining.
2.11 Quantification of changes in p21 and p27 expression
To assess the effect of eNOS overexpression on p21 and p27 expression we performed densitometry. The densitometry after serum stimulation is expressed relative to the quiescent state. The data were calculated on three separate experiments.
2.12 Detection of apoptotic cells
For assessment of nucleosomal laddering, DNA was isolated from transduced cells 24 and 72 h after transduction and fractionated on 2% agarose gels as previously described [18].
PCSMCs were plated in a 60x15 mm dish and transduced as described above. Cells were harvested by trypsinization at 24 or 72 h after transduction. The cells were pooled with their culture medium so cells that had lost their adherent properties during apoptosis (‘floaters’) were included in the analysis. Cells were pelleted, washed and resuspended in 400 µl binding buffer (BB, 100 mM HEPES (pH 7.4), 1.5 M NaCl, 50 mM KCl, 10 mM MgCl 2, 18 mM CaCl2) and 100 µl (1x106 cells) aliquoted to 4-ml Falcon tubes for labeling and FACS analysis. Cells were incubated with 1 µg/sample annexin-V–biotin conjugate (Trevigen, Gaitherburg, MD, USA) at 4°C in the dark for 20–30 min, washed and fluorescently labeled with streptavidin–phycoerythrin (PE) (Molecular Probes, Eugene, OR, USA) at 1 µg per sample under the same conditions. Labeled cells were washed and resuspended in 400 µl BB containing 6 µg/ml 7-aminoactinomycin D (7-AAD, Molecular Probes) and 2% formalin (Sigma, St. Louis, MO, USA). Cells were analyzed on a fluorescence activated cell sorter (Facstar, Benton Dickinson) within 2 h of labeling. Data were analyzed using the PC LYSIS program (Benton Dickinson). FACS gating based on forward scatter and side scatter was used to exclude cellular debris and doublets so that typically 14 000±2000 out of 20 000 cells were selected for analysis. Every experiment included control samples, which had been transduced with Adβgal or exposed to diluent alone. Tunicamycin (6 mmol/l) was used as a positive control for apoptosis. The experiment was repeated on three (Day 1) or six (Day 3) separate occasions and the data analyzed by ANOVA with Fisher's test.
2.13 Statistics
Data are presented as mean±S.E. Statistical analysis was performed by ANOVA to detect significant differences in multiple comparisons. An unpaired Student's t test was used to detect significant differences when two groups were compared. A value of P<0.05 was considered to be statistically significant.
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3 Results |
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3.1 Assessment of eNOS expression in vascular SMCs
Transgene expression was sought in cells transduced with AdeNOS by immunohistochemistry, NADPH diaphorase staining and western blot analysis. Control cells were transduced with an equivalent titer of AdβGal or were exposed to diluent alone. Transgene expression was determined 48 h after transduction. eNOS immunoreactivity was not detected in AdβGal- or non-transduced cells. In contrast, cells transduced with AdeNOS displayed positive immunoreactivity for eNOS (Fig. 1). In addition, expression of recombinant eNOS in transduced PCSMCs was confirmed by NADPH diaphorase staining. In the presence of βNADPH, NOS reduces NBT to formazan, which appears as a dark blue cytosolic stain. Such staining was present in AdeNOS-transduced cells but not in non-transduced cells or AdβGal transduced cells (Fig. 2). Finally, Western blot analysis demonstrated recombinant eNOS expression only in AdeNOS-transduced cells. eNOS expression was demonstrated in AdeNOS and not AdβGal transduced cells (Fig. 3). Thus, eNOS expression was demonstrated by three independent means in PCSMCs transduced with the adenoviral vector encoding eNOS.
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3.2 Functional assessment of recombinant protein expression
NO stimulates soluble guanylate cyclase to generate cGMP. Thus, functional protein expression was examined by measurement of cGMP in the basal and stimulated states (Fig. 4). Basal cGMP levels were significantly increased in AdeNOS compared to Adβgal- and non-transduced cells (753.5±286.6 vs. 5.7±12.8 vs. 2.1±4.8 pmol/mg protein, P<0.01). Moreover, stimulation of AdeNOS transduced cells by calcium ionophore (1 µmol/l) and L-arginine (0.1 mmol/l), resulted in a significant increase in cGMP generation (1992.6±1489.2 pmol/mg protein). In contrast, this was not observed in control cells.
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3.3 Cell cycle analysis of PCSMCs by flow cytometry
In the quiescent state (serum-deprived), non-transduced, Ad βGal- or AdeNOS-transduced cells displayed 89.6±2.6, 90.4±5.2 and 88.6±6.4%, respectively, in the G0/G1 phase of the cell cycle. After 24 h of serum stimulation, 45.9±10.8% of non-transduced cells and 38.8±8.1% of AdβGal-transduced cells had progressed into G2/M+S phases of cell cycle, and only 54.1±10.7 and 61.2±8.1%, respectively, remained in G0/G1. In contrast, 74.6±1.6% of AdeNOS transduced cells remained in the G0/G1 phase of the cell cycle, and only 25.4±1.6% had progressed into G2/M+S phases of cell cycle. (P=0.001 for AdeNOS vs. non-transduced, and P=0.0173 for Ad βGal vs. AdeNOS transduced cells). Thus, AdeNOS transduction resulted in a delay in cell cycle progression (Fig. 5) (Table 1).
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3.4 Upregulation of p27 and p21 expression after eNOS gene transfer
Quiescent control and AdβGal-transduced cells demonstrated high levels of p27 and p21 expression. After 24 h of serum stimulation, p27 and p21 expression substantially decreased in both sets of control cells. In contrast, p27 and p21 expression did not significantly decrease in AdeNOS-transduced cells (Figs. 6 and 7
). Using densitometric analysis there was no difference in p21 or p27 expression in quiescent cells. p21 and p27 expression was increased in stimulated eNOS transduced cells compared to diluent exposed and Adβgalactosidase transduced cells (Figs. 6b and 7b).
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3.5 eNOS gene transfer does not induce apoptosis
Nucleosomal laddering was not detected in AdeNOS transduced or control cells (data not shown).
Twenty-four hours following transduction, 9.0±4.2% of non-transduced cells, 7.9±2.5% of AdβGal transduced cells and 12.1±6.1% of AdeNOS transduced cells displayed evidence of apoptosis. There was no difference between the groups. In contrast, 61.9±21.8% of cells exposed to tunicamycin displayed evidence of apoptosis. The percentage of necrotic cells was similar in the four groups. Seventy-two hours after transduction, the percentage of apoptotic cells was not increased by eNOS gene transfer (7.5±2.5% of non-transduced cells, 13.6±2.8% of Ad βGal transduced cells, and 13.9±3.3% of AdeNOS transduced cells, respectively were apoptotic). As with day 1, there was a marked increase in apoptotic cells in the group exposed to tunicamycin (75.0±10.6%). The percentage of necrotic cells in each group was similar. Thus, AdeNOS transduction did not induce apoptosis (Fig. 8).
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4 Discussion |
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In this study, we have demonstrated that gene transfer of eNOS to vascular SMCs leads to functional recombinant protein expression resulting in a delay in cell cycle progression, associated with upregulation of the cyclin dependent kinase inhibitors p27 and p21. In contrast, induction of apoptosis was not observed in AdeNOS transduced cells. This suggests that adenoviral mediated gene transfer of eNOS to vascular SMCs inhibits cell proliferation via inhibition of cell cycle progression due to up regulation of p27 and p21 and that apoptosis is not involved.
SMC proliferation plays a pivotal role in a number of disease states characterized by vascular proliferation including atherosclerosis [19] and restenosis [20]. A greater understanding of the molecular defects underlying these conditions may result in more rational therapeutic approaches. NO donors have been shown to inhibit SMC proliferation [4] but systemic delivery of these agents has not reduced the occurrence of post-angioplasty restenosis. The use of NO donors is limited by systemic side effects such as hypotension. In contrast, local delivery of the gene for NOS may increase vascular wall NO generation at the site of injury and thus avoid systemic side effects associated with the use of NO donors. NOS gene transfer has other attractive features including the pleiotropic effects of NO with potential beneficial effects on a number of processes implicated in intimal hyperplasia. The potential use of eNOS gene transfer in vascular disease has recently been reviewed [21]. We have previously shown that adenoviral-mediated gene transfer of eNOS to vascular SMCs inhibits cell proliferation in vitro [10]. The mechanism of this effect was not studied but may include delayed cell cycle progression or induction of apoptosis.
Transit through G1 of the cell cycle requires activation of cyclin–CDK complexes [22,23]. Cyclin dependent kinase inhibitors (CKI) such as p27 and p21 inhibit these complexes. Expression of these proteins in vascular SMCs, porcine arteries and human coronary arteries has recently been examined [24]. p27 and p21 are expressed in quiescent SMCs and downregulated following serum stimulation. Furthermore, results from that study suggested that p27 contributes to the remodeling process in vascular diseases by the arrest of vascular SMCs in the G1 phase of the cell cycle. In the current study, eNOS expression in vascular SMCs delayed progression through the cell cycle in comparison to cells transduced with a control adenoviral vector or exposed to diluent alone. In contrast, there was no evidence of a delay in cell cycle progression in AdβGal-transduced cells. This is important, as adenoviral vectors have recently been shown to delay progression through the cell cycle in a bronchial cell line [25].
The mechanism of the delay in cell cycle progression was next studied. As progression through the cell cycle is regulated by CKIs, we sought to examine the effect of eNOS gene transfer on expression of p27 and p21. In the current study, we demonstrate that eNOS gene transfer to vascular SMCs results in upregulation of p27 and p21. Overexpression of p21, has been shown to limit intimal cell proliferation in response to injury [26]. As NO has many other potentially beneficial effects, eNOS gene transfer may have some advantages over CKI gene transfer in the management of disorders characterized by intimal hyperplasia. NO donors have previously been shown to upregulate p21 and have no effect on p27 [14]. The difference in results obtained in that experiment compared to ours is not immediately obvious but may include the fact that the biological effects resulting from NO generated as a result of NO donors or NOS gene transfer may not be directly comparable. In addition, the current experiments were performed in vitro and therefore caution should be exercised prior to extrapolation to in vivo events.
It has been suggested that vascular remodeling involves a balance between cell proliferation and apoptosis. Apoptosis has been detected in atherosclerotic [27] and restenotic [28] lesions. The autocrine and paracrine factors influencing the balance between apoptosis and cell division were studied by Pollman et al. [17]. They observed that SMC apoptosis was promoted by NO and inhibited by angiotensin. We were unable to detect an increase in apoptosis in vascular SMCs transduced with eNOS. The reason for the discrepancy is not clear but may involve differences in the amount of NO generated via NO donor versus gene transfer of eNOS. Furthermore, NO generated from NO donors or NOS gene transfer may have different biological effects due to differences in time course of NO generation and the additional effects of other metabolic products of the NO donors. In addition to the source of NO, the effect of the NOS isoform is also of interest. Recently, liposome-mediated iNOS overexpression in vascular SMCs was found to induce apoptosis [29]. In contrast, in our study, adenoviral vectors were used to transfer the gene for eNOS to vascular SMCs and in spite of highly efficient gene transfer, there was no evidence of apoptosis induction. This suggests that the effect of NOS gene transfer may vary depending on the isoform studied.
There is now evidence from a number of sources that transfer of eNOS or iNOS gene results in reduced intimal hyperplasia after injury [11,12,26,30]. The mechanism underlying this effect may vary depending on the NOS isoform studied. Our report suggests that delay in cell cycle progression may be of more importance in eNOS-mediated inhibition of SMC proliferation. In contrast, while the effect of iNOS gene transfer on cell cycle progression has not been reported, there is evidence that induction of apoptosis occurs following iNOS gene transfer. Thus, NO derived from different NOS isoforms may inhibit cell proliferation via distinct mechanisms.
In conclusion, we have reported that eNOS gene transfer to vascular SMCs results in a delay in cell cycle progression associated with upregulation of p27 and p21 and does not induce apoptosis. A direct comparison of the effects of eNOS and iNOS gene transfer may shed light on which is the isoform of choice in gene therapy approaches to vasculoproliferative disorders.
Time for primary review 19 days.
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Acknowledgements |
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This work was supported by Mayo Clinic intramural research grants (T.O.B.), the Bruce and Ruth Rappaport Program in Vascular Biology, National Institutes of Health grants HL-44116, HL-53542 (Z.S.) and HL-58080 (T.O.B.). J.S. is supported by grants from Novo Nordisk and Tokyo Medical University.
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