Expression and Function of Recombinant Endothelial NO Synthase in Coronary Artery Smooth Muscle Cells
Abstract Smooth muscle cells (SMCs) play a key role in the pathogenesis of vascular diseases. The objectives of this study were to determine whether transfer of recombinant endothelial nitric oxide synthase (eNOS) gene to porcine coronary artery smooth muscle cell (CSMCs) would result in expression of a functional enzyme and to assess the effect of expression of eNOS on cell proliferation. CSMCs were transduced in vitro with adenoviral vectors encoding cDNA for eNOS (AdeNOS) and β-galactosidase (AdβGal). In contrast to AdβGal- or sham-transduced cells, CSMCs transduced with AdeNOS stained positive with the NADPH-diaphorase stain, acquired calcium-dependent NOS activity (measured by the conversion of [3H]l-arginine to [3H]l-citrulline), had increasing cyclic 3′,5′ cGMP levels with increasing concentrations of the vector, and produced increased amounts of nitrite. cGMP production by AdeNOS-transduced cells was augmented by increasing intracellular levels of the eNOS cofactor tetrahydrobiopterin. CSMCs transduced with AdeNOS showed diminished serum-stimulated DNA synthesis as measured by thymidine uptake. Cell proliferation was diminished in AdeNOS-transduced CSMCs as assessed by cell counts 3 and 6 days after serum stimulation of quiescent CSMCs. The present study demonstrates that adenovirus-mediated gene transfer of eNOS to CSMCs results in the expression of a functional enzyme whose activity can be augmented by increasing intracellular levels of tetrahydrobiopterin. Expression of recombinant eNOS in CSMCs results in inhibition of serum-stimulated DNA synthesis and cell proliferation. These findings imply that eNOS gene transfer to SMCs may be a unique mode of increasing local NO production in the arterial wall.
Presented in part at the 69th Scientific Sessions of the American Heart Association, New Orleans, La, November 14-17, 1996, and previously published in abstract form in Circulation. 1996;94(suppl I):I-146.
- Received August 22, 1996.
- Accepted April 15, 1997.
Smooth muscle cells form the principal cellular component of arteries and veins and play a key role in the formation of a neointima in various vascular diseases, including atherosclerosis, restenosis, vein graft disease, and transplant vasculopathy.1 Several pharmacological agents limit neointimal formation after balloon injury in experimental models, but none has been effective in clinical trials.2 This is likely due to the more complex vascular lesions in humans that involve several pathogenetic mechanisms, including extracellular matrix secretion, inflammation, and platelet-fibrin thrombus formation.3 4 Consequently, agents with pleiotropic actions are more likely to be effective in limiting neointimal formation rather than those that act on a single pathogenetic mechanism.5 NO is a simple diatomic molecule that plays an important role in regulating vascular tone and preventing thrombosis. In addition, NO donors inhibit SMC proliferation,6 migration,7 matrix production,8 9 and platelet10 and monocyte adhesion.11 These factors are important in determining neointimal formation, particularly after balloon angioplasty or stenting. Increasing the local production of NO may have a unique role in inhibiting formation of a neointima in vascular diseases.12
NO is a highly reactive gas with a short half-life and limited solubility in aqueous media, making local delivery of NO to the vessel wall difficult.13 Transfer of the eNOS gene to the arterial wall has been shown to increase local NO production.14 The aim of the present study was to characterize, at the cellular level, adenovirus-mediated gene transfer of eNOS to porcine CSMCs in vitro. Adenoviral vectors mediate efficient gene transfer to a wide variety of cells and tissues15 and may allow site-specific delivery of recombinant proteins to the vessel wall as well as genetic engineering of vascular wall cells.16 We sought to (1) determine whether gene transfer of eNOS to CSMCs would result in expression of a functional enzyme; (2) study the effects of increased availability of tetrahydrobiopterin, a cofactor for eNOS, on recombinant enzyme activity; and (3) assess whether the expression of recombinant eNOS would result in a biological effect, namely, inhibition of cell proliferation.
Construction, Propagation, and Purification of Adenoviral Vector
Recombinant adenovirus containing cDNA encoding eNOS was generated as described.17 In brief, bovine eNOS cDNA (provided by Dr David Harrison, Emory University, Atlanta, Ga) was cloned into the pACCMVpLpA vector (provided by Dr Robert Gerard, University of Texas Southwestern Medical Center, Dallas, Tex). The resulting plasmid was linearized and cotransfected with dl309 into 293 cells by calcium phosphate/DNA coprecipitation. dl309 is a biologically selected, restriction enzyme–site-loss variant of wild-type adenovirus.18 293 cells are human embryonic kidney carcinoma cells that have been transformed with the left end of human adenovirus type 5 DNA.19 Recombinant adenoviral vectors encoding eNOS were generated by homologous recombination.17 Viral plaques were picked and propagated in 293 cells. Viral DNA was enriched by Hirt extraction20 and screened by restriction mapping and PCR for the presence of eNOS cDNA. Positive plaques underwent two additional rounds of plaque purification in 293 cells. Stock solutions were prepared from positive plaques and these were used to generate high-titer preparations. Viral preparations were produced by infecting confluent monolayers of 293 cells in 175-cm2 flasks with viral stock at an MOI of 1 to 10. Virus was purified by double CsCl gradient ultracentrifugation and dialyzed against 10 mmol/L Tris, 1.0 mmol/L MgCl2, 1.0 mmol/L HEPES, and 10% glycerol for 4 hours at 4°C. Viral titer was determined by plaque assay.17 eNOS activity was confirmed by positive NADPH diaphorase staining21 in confluent 293 cells transduced with AdeNOS. The defective nature of AdeNOS for replication was tested by infecting human embryonic lung cell diploid cell cultures. Replication-competent viruses at an MOI ≥10 produced cytopathic effects and destroyed the monolayer in <3 days. Infection with AdeNOS at a comparable MOI produced no observable cytopathic effects after 5 days. A recombinant replication-defective adenoviral vector encoding the Escherichia coli AdβGal gene driven by the cytomegalovirus promoter22 was obtained from Dr James Wilson (University of Pennsylvania, Philadelphia) and used as a control. It was propagated, isolated, and quantified as described above.
DMEM, medium M199, heat-inactivated FCS, HBSS, PBS, penicillin, streptomycin, l-glutamine, and 0.25% trypsin-EDTA solution were purchased from GIBCO Laboratories. [3H]Thymidine was obtained from New England Nuclear Research Products; [3H]l-arginine was purchased from Amersham; sepiapterin was purchased from Alexis Corp; leupeptin and collagenase were obtained from Boehringer Mannheim. All other reagents, unless specified otherwise, were obtained from Sigma Chemical Co.
Porcine CSMCs were obtained from the coronary arteries of domestic crossbred pigs by enzymatic digestion with 1% collagenase. 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 minutes. 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 FCS, l-glutamine (2 mmol/L), penicillin (100 U/mL), streptomycin (100μg/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 replanted in 75-cm2 flasks. Identity of the cells was confirmed by the typical “hill-and-valley” appearance at confluence and positive smooth muscle α-actin staining. The cells were kept at 37°C in humidified 5% CO2/95% O2. CSMCs were passaged by trypsinization, and cells between the third and seventh subpassages were used for the experiments.
Transduction With Adenoviral Vectors
CSMCs were plated at a density of 1 to 2 ×104/cm2 in 6- or 24-well plates (Corning Glass Works) and cultured overnight in medium M199 with 10% FCS. The cells were transduced with adenoviral vectors the next day. Cells were washed with PBS, and the vector solution in PBS with 0.5% albumin was added. Adenoviral vector concentration was quantified as MOI, which indicates the number of vector particles per cell. Cells were exposed to the vector solution for 1 hour. The vector solution was aspirated, cells washed with PBS, and the medium replaced.
The NADPH-diaphorase reaction localizes the presence of NOS activity in tissue.21 Forty-eight hours after transduction with vectors at increasing MOIs, cells were washed with PBS and then fixed in 4% p-formaldehyde for 5 minutes. 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 minutes at 37°C. The solution was replaced with PBS, and the cells were assessed for staining by phase-contrast microscopy.
NOS Enzyme Activity
NOS enzyme activity was measured 48 hours after transduction of confluent layers of CSMCs in 75-cm2 flasks with AdeNOS or AdβGal at an MOI of 200. The assay is based on the biochemical conversion of l-arginine to l-citrulline by NOS.23 In brief, cells were trypsinized and centrifuged to obtain a cell pellet. The pellet was resuspended in cold lysis buffer (0.32 mmol/L sucrose, 50 mmol/L Tris HCl, 0.1 mmol/L EDTA, 100 μg/mL PMSF, 10 μg/mL leupeptin, 10 μg/mL pepstatin A, and 10 μg/mL antipapain, pH 7.8), sonicated, and kept on ice. To analyze NOS activity, cell lysates (150 to 250 μg) were incubated with 1 mmol/L NADPH, 10 μmol/L tetrahydrobiopterin, 0.83 mmol/L CaCl2, 5 μmol/L [3H]l-arginine, 54 mmol/L l-valine, 1.2 mmol/L MgCl2, 2 μmol/L FAD, and 50 U/mL calmodulin for 1 hour at 25°C. Calcium-independent NOS activity was measured by replacing CaCl2 with 1 mmol/L EGTA in replicate samples. Replicates were also incubated with 2 mmol/L l-NMMA to assess the degree of inhibition of NOS activity. Incubations were terminated by adding ice-cold stop buffer (20 mmol/L HEPES, 8 mmol/L EDTA, pH 5.5). The reaction mix was then passed through Poly-Prep chromatography columns (Bio-Rad Laboratories) containing 1 mL of Dowex AG50W-X8 resin (Bio-Rad) and eluted with 4 mL of distilled water. Radioactivity in the flow-through was due to [3H]l-citrulline and was measured by scintillation spectroscopy. The protein concentration of cell lysates was measured by the method of Lowry et al.24
Measurement of cGMP
CSMCs were plated and transduced with AdeNOS or AdβGal at increasing MOIs as described above. cGMP levels in CSMCs were measured 48 hours later as follows. The medium was aspirated, and cells were incubated at 37°C in a control solution containing 1 mmol/L isobutyl methylxanthine to inhibit degradation of the cyclic nucleotides by phosphodiesterases. cGMP was extracted under acidic conditions by adding 0.1N HCl for 30 minutes and measured by radioimmunoassay (Amersham). To assess their effects on cGMP production, sepiapterin (10−4 mol/L), SOD (60 U/mL), and l-NMMA (10−4 mol/L) were added to the medium of cells transduced at an MOI of 200. In certain experiments, calcium ionophore (1 μmol/L) was added to the medium of transduced cells for 30 minutes prior to cGMP extraction. cGMP levels were normalized to the protein content of each well, which was estimated by the method of Lowry et al.24
Measurement of Nitrite
NO production was evaluated by the measurement of nitrite by spectrofluorometric assay.25 CSMCs were transduced with AdeNOS or AdβGal at an MOI of 200. Forty-eight hours later, the medium was aspirated and replaced with 2 mL of Krebs’ solution containing l-arginine (10−4 mol/L) and the calcium ionophore A23187 (10−6 mol/L). Cells were incubated at 37°C for 2 hours. The Krebs’ solution was aspirated and nitrite measured by reaction with 2,3-diaminonapthalene under acidic conditions to form 1-(H)-naphthotriazole, a fluorescent product. Formation of 1-(H)-naphthotriazole was measured by a spectrofluorometer (SLM-8000, Spectronic Instruments Inc) with an excitation wavelength of 365 nm and emission wavelength of 410 nm. NaOH (1N) was added to solubilize the cells for protein estimation by the method of Lowry et al.24 Nitrite levels were normalized to the protein content of each well.
Measurement of Tetrahydrobiopterin Levels
CSMCs were grown with or without sepiapterin (10−4 mol/L), a precursor of tetrahydrobiopterin, in 75-cm2 flasks for 48 hours. The cells were then trypsinized and centrifuged to obtain a cell pellet. The pellet was washed three times in PBS and stored at −80°C until further analysis. Measurement of tetrahydrobiopterin was performed by high-performance liquid chromatography after oxidation by MnO2 or iodine as previously described.26
Expression of GTP Cyclohydrolase-I mRNA in CSMCs
RT-PCR was used to detect expression of GTP cyclohydrolase mRNA in CSMCs. Total RNA was extracted from CSMCs with use of the RNA stat-60 kit (Tele-Test “B”/Inc), and integrity of the isolated RNA was confirmed by denaturing agarose gel electrophoresis. The RNA template (1 μg) was reverse transcribed using random hexamers and SuperScript II reverse transcriptase (GIBCO BRL). The cDNA was amplified by PCR using synthetic primers derived from conserved regions of human, chicken, rat, and mouse GTP cyclohydrolase sequences. oligo 4.0 software was used to design primers and determine optimal annealing temperature. Primers used were the following: upper 20-mer, 5′-TAC CAG GAG ACC ATC TCA GA-3′; lower 20-mer, 5′-CGC ATT ACC ATA CAC ATG TG-3′. PCR amplification was performed in a 25-μL reaction mixture containing 1 μL of cDNA, 10 pmol of each primer, 2 U of Taq DNA polymerase (GIBCO BRL), 2.5 μL of 10× buffer provided with the polymerase, and 2.0 mmol/L MgCl2. Each amplification cycle (40 cycles in all) consisted of denaturation at 94°C for 30 seconds, annealing at 59°C for 60 seconds, and elongation at 72°C for 30 seconds. PCR products were electrophoresed on a 1.5% agarose gel containing ethidium bromide and visualized by UV fluorescence. The PCR product was sequenced by using the upper primer of the PCR reaction. The sequence obtained revealed complete homology to conserved GTP cyclohydrolase regions within human, mouse, rat, and chicken sequences.
The effect of eNOS gene transfer on DNA synthesis was studied by [3H]thymidine incorporation. Cells were plated and transduced at MOIs of 10, 25, 50, 100, and 200 as described above in 24-well plates. After transduction, medium M199 containing 0.5% FCS was added for 48 hours to render the CSMCs quiescent. Cell growth was stimulated by replacing the medium and 0.5% FCS with fresh medium containing 10% FCS. Twenty hours later, 1 μCi of 3H-labeled thymidine was added per well and the cells incubated for 4 hours at 37°C. The medium was aspirated and cells washed with ice-cold PBS. Acid-insoluble material was precipitated with 10% trichloroacetic acid at 4°C and DNA extracted with 0.5N NaOH. The radioactivity was determined by scintillation spectroscopy.
For cell proliferation studies, cells were plated and grown overnight in medium M199 with 10% FCS. Cells were transduced at an MOI of 200 and rendered quiescent as described above. Cell proliferation was stimulated by replacing the medium containing 0.5% FCS with fresh medium containing 10% FCS. The culture medium was changed every 48 hours. Cell morphology was assessed daily by phase-contrast microscopy. In certain experiments, cell counts in the supernatant media were performed daily to quantify cell detachment. Cells were counted in a Coulter counter (model ZM, Coulter Electronics Ltd) prior to addition of medium M199 and 10% FCS on day 0, day 3, and day 6. Cell suspensions from certain wells were incubated with 0.4% trypan blue for 10 minutes to assess cell viability.
All experiments were performed on two to six replicates. Data are presented as mean±SEM. Statistical analysis was performed by ANOVA, followed by Scheffe’s test or unpaired Student’s t test as appropriate. A value of P<.05 was considered statistically significant.
Confirmation of Gene Transfer by NADPH- Diaphorase Staining
Expression of recombinant eNOS in transduced CSMCs was confirmed by NADPH diaphorase staining (Fig 1⇓). 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 (Fig 1C⇓) but not in AdβGal-transduced (Fig 1B⇓) or sham-transduced (Fig 1A⇓) cells. Increasing intensity of staining was observed with increasing MOIs in AdeNOS-transduced CSMCs (not shown). Similarly, expression of recombinant β-Gal was confirmed by X-Gal staining (not shown).
NOS Enzyme Activity
In contrast to AdβGal- or sham-transduced cells, NOS enzyme activity was present in AdeNOS-transduced cells as quantified by conversion of [3H]l-arginine to [3H]l-citrulline. This activity was nearly abolished by EGTA (1 mmol/L) and also by l-NMMA (2 mmol/L) (P<.001) (Fig 2⇓).
Effect of AdeNOS Vector Transduction on cGMP Levels
Increasing levels of cGMP were present in CSMCs 48 hours after transduction with the AdeNOS vector at increasing MOIs. cGMP levels were significantly lower (P<.01) in AdβGal-transduced cells and did not show a concentration-response effect (Fig 3⇓). l-NMMA (10−4 mol/L) added at the time of transduction significantly reduced cGMP levels in CSMCs transduced with the AdeNOS vector (P<.01) (Fig 4⇓). Exposure of the cells to the calcium ionophore A23187 (1 μmol/L) for 30 minutes, 48 hours after transduction, caused a 35±3% increase in cGMP levels of AdeNOS-transduced cells (P<.01), whereas no such effect was seen in AdβGal-transduced cells (data not shown).
In the presence of the calcium ionophore and l-arginine, nitrite production 48 hours after transduction was significantly greater (P<.01) in AdeNOS-transduced cells than in cells transduced with AdβGal (Fig 5⇓).
Effect of Sepiapterin on Tetrahydrobiopterin Levels and of Sepiapterin and SOD on cGMP Levels
Addition of sepiapterin (10−4 mol/L) to the medium caused a marked increase in tetrahydrobiopterin levels of CSMCs (5093±184 versus <0.7 pmol/mg protein, P<.0001; data from two experiments in triplicate). When sepiapterin was added to AdeNOS-transduced cells, significant augmentation was seen in NO production as measured by cGMP levels. Addition of SOD alone or in combination with sepiapterin also increased cGMP levels in AdeNOS-transduced cells (Fig 6⇓). Sepiapterin or SOD had no effect on cGMP levels in AdβGal- or sham-transduced cells (data not shown).
GTP Cyclohydrolase-I Expression
Specific mRNA for GTP cyclohydrolase I was detected in porcine CSMCs by RT-PCR. All PCR products were found to be of the predicted size on agarose gels (Fig 7⇓). Identical results were obtained in two different experiments. The specificity of the PCR reaction was confirmed by sequencing PCR products. The sequence of the 323-bp PCR product showed >98% homology to the conserved sequences of GTP cyclohydrolase-I from four different species.
Effect of AdeNOS and AdβGal Transduction on Thymidine Uptake
At an MOI of 10, thymidine uptake was not different in AdeNOS-transduced CSMCs when compared with AdβGal- and sham-transduced cells. At higher MOIs, thymidine uptake was significantly inhibited in AdeNOS-transduced CSMCs compared with AdβGal- and sham-transduced cells (P<.01), the degree of inhibition being proportional to the MOI (Fig 8⇓). At an MOI of 200, a significant decrease in uptake by AdβGal-transduced cells in comparison with sham-transduced cells was noted (P<.05).
Effect of AdeNOS and AdβGal Transduction on Cell Proliferation
Cell counts after transduction and quiescence and prior to serum stimulation (day 0) were similar among the three groups (Fig 9⇓). CSMCs transduced with AdeNOS showed a significant decrease in cell counts compared with AdβGal- and sham-transduced cells on days 3 and 6 after stimulation with medium M199 and 10% FCS (Fig 9⇓). Cell viability as measured by trypan blue exclusion was >95% in all three groups; cell counts in supernatants performed daily after serum stimulation were not significantly different among the three groups (data not shown).
Formation of a neointima in vascular disease states is to a large extent secondary to vascular SMC migration, proliferation, and matrix secretion.1 The SMC is therefore an important target of various systemic and local therapies to limit neointimal formation.12 Expression of recombinant proteins in vascular SMCs may allow control of local vascular function and also serve as a tool in the study of vascular biology.16 Expression of recombinant eNOS in vascular SMCs has unique potential for influencing vascular disease states owing to the effects of NO on vasomotor tone, platelet adhesion/aggregation, monocyte adhesion, and cell proliferation. In this study, we have demonstrated that expression of recombinant eNOS in porcine CSMCs results in a functionally active enzyme with resulting increases in cGMP levels and nitrite production. The activity of the recombinant enzyme is augmented by increasing intracellular levels of tetrahydrobiopterin, a cofactor for eNOS. Most important, expression of recombinant eNOS resulted in inhibition of SMC DNA synthesis and cell proliferation.
Expression of recombinant eNOS was demonstrated by NADPH-diaphorase staining in transduced CSMCs. NADPH diaphorase and NOS activities are different properties of the same enzyme,21 and NADPH-diaphorase activity can be used as a marker for NOS.27 The functional activity of recombinant eNOS was demonstrated by the l-arginine to l-citrulline conversion assay, which directly measures NOS enzymatic activity. In contrast to AdβGal- and sham-transduced CSMCs, AdeNOS-transduced CSMCs exhibited significantly greater NOS activity, which was inhibited by l-NMMA. These observations are further supported by the increased cGMP and nitrite production by CSMCs transduced with AdeNOS. Nitrite is a stable end product of NO, and cGMP is formed by hydrolysis of GTP after activation of a soluble guanylate cyclase by NO.28 Both nitrite and cGMP levels are therefore reliable indicators of NO production by recombinant eNOS. The increase in cGMP levels induced by recombinant eNOS was inhibited by l-NMMA. Additionally, calcium ionophore A23187, a known stimulant of eNOS, significantly increased cGMP levels in AdeNOS-transduced cells, with no such effect on control cells.
Recent reports suggest that adenovirus-mediated gene transfer to the endothelium in vivo29 and to endothelial cells in vitro30 may lead to changes in cellular phenotype as well as increased production of cytokines. Therefore, a theoretical possibility exists that adenovirus-mediated gene transfer to CSMCs may result in expression of the cytokine iNOS isoform. Since AdβGal- or sham-transduced CSMCs did not stain positive with the NADPH-diaphorase stain, the iNOS isoform was unlikely to be present in these cells, as the stain does not differentiate among the various NOS isoforms. Furthermore, in sham- or AdβGal-transduced CSMCs, increases in NOS enzymatic activity or cGMP levels were not observed. In the AdeNOS-transduced CSMCs, NOS enzymatic activity was nearly abolished by the calcium chelator EGTA, indicating that the enzyme activity was calcium dependent and therefore not due to iNOS, which is calcium independent. These findings confirm the absence of iNOS activity in the vector- or sham-transduced cells.
Vascular SMCs do not produce NO in the resting state, but after exposure to cytokines, iNOS is expressed by these cells.31 iNOS produces large amounts of NO in a complex reaction that requires several cofactors, including tetrahydrobiopterin. Tetrahydrobiopterin availability is an absolute requirement for NO synthesis after iNOS induction in vascular smooth muscle,32 and GTP cyclohydrolase I, an enzyme that catalyzes the first step in the synthesis of tetrahydrobiopterin, is coinduced with iNOS in cytokine-treated SMCs.32 33 Our results demonstrate that basal levels of tetrahydrobiopterin in CSMCs were sufficient to support the activity of recombinant eNOS. Expression of GTP cyclohydrolase I mRNA in CSMCs as demonstrated by RT-PCR may result in the production of small amounts of tetrahydrobiopterin that allow recombinant eNOS to be functionally active.
The effect of increasing intracellular tetrahydrobiopterin levels on the enzymatic activity of recombinant eNOS was examined by adding sepiapterin. Sepiapterin, a precursor of tetrahydrobiopterin via a salvage pathway,32 caused a marked increase in tetrahydrobiopterin levels in CSMCs. Administration of sepiapterin to CSMCs transduced with AdeNOS resulted in a 75% increase in cGMP levels, indicating an increase in recombinant eNOS activity. Tzeng and coworkers34 were unable to document functional enzyme activity after retrovirus-mediated gene transfer of the iNOS gene to rat pulmonary artery SMCs unless the medium was supplemented with tetrahydrobiopterin. This may be due to the different NOS isoform and SMC type in their experiments. On the other hand, eNOS activity can be demonstrated in freshly isolated endothelial cell lysates,35 with a nearly twofold increase in enzyme activity after addition of tetrahydrobiopterin to the medium. Our results are similar, demonstrating a functional recombinant eNOS in CSMCs and an increase in intracellular tetrahydrobiopterin resulting in augmented eNOS enzyme activity.
A significant increase in cGMP levels was also seen in CSMCs transduced with AdeNOS after addition of SOD to the culture medium. This is likely due to the “protection” of NO (produced by recombinant eNOS) from the superoxide radical, which can inactivate NO.36 37 The redox state in the cell or its environment may influence the bioactivity of NO, and increasing antioxidant capacity may be a method of potentiating the effects of NO produced by recombinant eNOS.
Gene transfer and functional activity of recombinant eNOS resulted in inhibition of serum-stimulated proliferation of porcine CSMCs. Both DNA synthesis, as assessed by thymidine uptake, and cell growth, as demonstrated by cell counts, were diminished in AdeNOS-transduced cells. No effect on DNA synthesis was noted in cells transduced with the AdeNOS vector at an MOI of 10, despite a significant elevation of cGMP levels compared with that in AdβGal-transduced cells. These results suggest that NO-induced cytostasis requires high concentrations of NO. The antimitogenic effect secondary to recombinant eNOS expression was not due to cell detachment, as cell counts from the media were not different among the three groups. Cell viability as estimated by trypan blue exclusion was >95% in all three groups, suggesting that the titer of the vector used in this study was not associated with cytotoxicity. A small but significant decrease in thymidine uptake was observed in cells transduced with the AdβGal vector at an MOI of 200 compared with sham-transduced cells. This indicates that adenoviral vectors may induce changes in cell function that are separate from those of the transgene. Because different viral sources for generation of recombinant adenoviral vectors could potentially result in different changes in cellular function, it is important that the control vector and the vector containing the transgene of interest be closely matched. The control vector used in this study was E1-E3 deleted,22 whereas the AdeNOS vector was E1 deleted. However, because E3 transcription is dependent on E1a expression, an E1a/E3–deleted virus should behave similarly to an E1a-deleted vector, thereby enabling investigators to distinguish effects of the transgene from nonspecific effects of the vector.
Owing to NO’s labile and reactive nature, most in vitro and in vivo studies of this compound rely on specific pharmacological tools, such as NO-donor compounds, rather than authentic NO. Although NO-releasing compounds have been shown to inhibit SMC proliferation in vitro, the effect is seen only at high pharmacological doses in the millimolar range.6 Continuous delivery of NO may be needed to cause a cytostatic effect, as NO donors effect cell proliferation only when a given molar quantity is administered slowly over a prolonged time rather than in large doses for a brief time.38 Furthermore, the use of NO donors in the clinical setting is complicated by the need for metabolic activation in some instances,39 tolerance after repeated treatment,40 and hypotension at higher doses. Our observations indicate that adenovirus-mediated gene transfer of eNOS to CSMCs results in a functionally active recombinant enzyme, which may provide a continuous supply of NO for the duration of transgene expression. This technique may serve as a method of local NO delivery that overcomes some of the disadvantages of NO donors for use in site-specific vascular wall therapy.
In summary, the present study demonstrates functional eNOS activity after adenovirus-mediated gene transfer of eNOS cDNA to porcine CSMCs in vitro. Enzymatic activity of eNOS-transduced cells was augmented by increasing intracellular levels of tetrahydrobiopterin. Finally, NO produced by the recombinant enzyme resulted in a biological effect in the form of inhibition of serum-stimulated CSMC proliferation. These findings may have important implications for vascular wall gene transfer of eNOS as well as cell-based therapies involving genetic engineering of vascular wall cells.
Selected Abbreviations and Acronyms
|AdeNOS||=||recombinant adenovirus encoding endothelial nitric oxide synthase gene|
|AdβGal||=||recombinant adenovirus encoding the Escherichia coli β-galactosidase gene|
|CSMC||=||porcine coronary artery smooth muscle cell|
|eNOS||=||endothelial nitric oxide synthase|
|MOI||=||multiplicity of infection|
|PCR||=||polymerase chain reaction|
|RT-PCR||=||reverse transcriptase–polymerase chain reaction|
The work was supported by Mayo Foundation intramural research grants (to T.O., R.S.S., V.J.P., and L.A.F.); the J. Holden DeHaan Foundation (to R.S.S.); and in part by National Institutes of Health grants HL-51736 (to L.A.F.), HL-44116, and HL-53532 (to Z.S.K.). The authors thank Justin Anderson, Adele Stelter, and Sharon Guy for invaluable technical assistance.
Schwartz RS, Srivatsa SS, Simari RD, Holmes DR Jr. Coronary restenosis: insights from animal models. In: Vetrovec GW, Carabello, BA, eds. Invasive Cardiology: Current Diagnostic and Therapeutic Issues. Armonk, NY: Futura Publishing Co Inc; 1996:67-76.
Wilcox JN. Thrombin and other potential mechanisms underlying restenosis. Circulation. 1991;84:432-435.
Garg UC, Hassid A. Nitric oxide generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest. 1989;83:1774-1776.
Sarkar R, Meinberg EG, Stanley JC, Gordon D, Webb RC. Nitric oxide reversibly inhibits the migration of cultured vascular smooth muscle cells. Circ Res. 1996;78:225-230.
Kolpakov V, Gordon D, Kulik TJ. Nitric oxide-generating compounds inhibit total protein and collagen synthesis in cultured vascular smooth muscle cells. Circ Res. 1995;76:305-309.
Mellion BT, Ignarro LJ, Ohlstein EGH, Pontecarvo EG, Hyman AI, Kadowitz PJ. Evidence for the inhibitory role of guanosine 3′:5′-monophosphate in ADP induced human platelet aggregation in the presence of nitric oxide and related vasodilators. Blood. 1981;57:946-955.
Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci U S A. 1991;88:4651-4655.
Gibbons GH, Dzau VJ. Molecular therapies for vascular diseases. Science. 1996;272:689-693.
von der Leyen HE, Gibbons GH, Morishita H, Lewis HP, Zhang L, Nakajima M, Kaneda M, Cooke JP, Dzau VJ. Gene therapy inhibiting neointimal vascular lesion: in vivo transfer of endothelial cell nitric oxide synthase gene. Proc Natl Acad Sci U S A. 1995;92:1137-1141.
Schneider MD, French BA. The advent of adenovirus: gene therapy for cardiovascular disease. Circulation. 1993;88:1937-1942.
Nabel EG. Gene therapy for cardiovascular disease. Circulation. 1995;91:541-548.
Spector DJ, Samaniego LA. Construction and isolation of recombinant adenovirus with gene replacements. Methods Mol Genet. 1995;7:31-44.
Graham FL, Smiley J, Russell WC, Nairn R. Characteristics of a human cell line transformed by human adenovirus type 5. J Gen Virol. 1977;36:59-74.
Hope BT, Michael GJ, Knigge KM, Vincen SR. Neuronal NADPH diaphorase is a nitric oxide synthase. Proc Natl Acad Sci U S A. 1991;88:2811-2814.
Yang Y, Raper SE, Cohn JA, Engelhardt JF, Wilson JM. An approach for treating the hepatobiliary disease of cystic fibrosis by somatic gene transfer. Proc Natl Acad Sci U S A. 1993;90:4601-4605.
Bredt DS, Snyder SH. Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum. Proc Natl Acad Sci U S A. 1989;86:9030-9033.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275.
Klimaschewski L, Kummer W, Mayer B, Couraud JY, Preissler U, Philippin B, Heym C. Nitric oxide synthase in cardiac nerve fibers and neurons of rat and guinea pig heart. Circ Res. 1992;71:1533-1537.
Arnold WP, Mittal CJK, Katusiki CK, Murad F. Nitric oxide activates guanylate cyclase and increases guanosine 3′:5′-cyclic monophosphate levels in various tissue preparations. Proc Natl Acad Sci U S A. 1977;74:3203-3207.
Newman KD, Dunn PF, Owens JW, Schulick AH, Virmani R, Sukhova G, Libby P, Dichek DA. Adenovirus-mediated gene transfer into normal rabbit arteries results in prolonged vascular cell activation, inflammation, and neointimal hyperplasia. J Clin Invest. 1995;96:2955-2965.
Rosenzweig A, Yoshida M, Dichek DA, Gimbrone MA. Adenoviral vectors affect human endothelial cell phenotype in vitro. Circulation. 1995;92(suppl I):I-296. Abstract.
Gross S, Levi R. Tetrahydrobiopterin synthesis: an absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle. J Biol Chem. 1992;267:25722-25729.
Rosenkranz-Weiss P, Sessa WC, Milstein S, Kaufman S, Watson CA, Pober JS. Regulation of nitric oxide synthesis by proinflammatory cytokines in human umbilical vein cells. J Clin Invest. 1994;93:2236-2243.
Ignarro LJ, Lippton H, Edwards JC. Mechanism of vascular smooth muscle relaxation by organic nitrates, nitroprusside and nitric oxide: evidence for the involvement of S-nitrosothiols as intermediates. J Pharmacol Exp Ther. 1981;218:739-749.
Needleman P, Johnson EM Jr. Mechanism of tolerance development to organic nitrates. J Pharmacol Exp Ther. 1973;184:709-715.