© 1997 American Heart Association, Inc.
Articles |
Expression and Function of Recombinant Endothelial NO Synthase in Coronary Artery Smooth Muscle Cells
From the Divisions of Cardiovascular Disease (I.J.K., R.S.S., V.J.P.), the Departments of Anesthesiology and Pharmacology (M.T., Z.S.K.) and of Endocrinology and Metabolism (L.A.F., T.O.), Mayo Clinic, Rochester, Minn; and the Laboratory of Cell Biology, National Institutes of Mental Health, National Institutes of Health (S.M.), Bethesda, Md.
Correspondence to Timothy O'Brien, MD, Senior Associate Consultant, Department of Endocrinology and Metabolism, Mayo Clinic, 200 First St SW, Rochester, MN 55905. E-mail obrien.timothy{at}mayo.edu
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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 ß-galactosidase (AdßGal). In contrast to AdßGal- 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.
Key Words: nitric oxide • gene transfer • nitric oxide synthase • adenovirus • vascular smooth muscle cells
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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.
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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 AdßGal 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.
Chemicals
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.
Cell Culture
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 
-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
x104/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.
NADPH-Diaphorase Stain
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 ßNADPH, 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
AdßGal 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 AdßGal 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 AdßGal 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 10x 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.
DNA Synthesis
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.
Cell Proliferation
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.
Statistics
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.
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Results |
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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 ßNADPH, 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 AdßGal-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 ß-Gal was confirmed by X-Gal staining (not shown).
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NOS Enzyme Activity
In contrast to AdßGal- 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).
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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 AdßGal-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 AdßGal-transduced cells (data not shown).
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Nitrite Production
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 AdßGal (Fig 5).
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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 AdßGal- or sham-transduced cells (data not shown).
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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.
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Effect of AdeNOS and AdßGal Transduction on Thymidine
Uptake
At an MOI of 10, thymidine uptake was not different in
AdeNOS-transduced CSMCs when compared with AdßGal- and
sham-transduced cells. At higher MOIs, thymidine uptake was
significantly inhibited in AdeNOS-transduced CSMCs compared with
AdßGal- 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 AdßGal-transduced cells in comparison with
sham-transduced cells was noted (P<.05).
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P<.05 vehicle vs
AdßGal.
Effect of AdeNOS and AdßGal 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 AdßGal-
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).
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Discussion |
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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 AdßGal- 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 AdßGal- 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 AdßGal-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 AdßGal-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 AdßGal 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.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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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.
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Footnotes |
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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.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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