1 Departments of Structural and Cellular Biology and 2 Pharmacology, Tulane University Health Sciences Center, New Orleans, Louisiana 70112
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ABSTRACT |
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Endothelial nitric oxide synthase (eNOS) is responsible for the production of nitric oxide (NO) in blood vessels. NO has been shown to be involved in the inhibition of vascular smooth muscle cell (VSMC) proliferation. In the present study, the eNOS gene was transferred into rat aortic smooth muscle cells by using an adenoviral vector, and the effect of endogenously produced NO on VSMC proliferation was investigated. The presence of eNOS in eNOS-transfected cells was confirmed by immunocytochemistry and Western blot analysis. eNOS transfection resulted in inhibition of VSMC proliferation. This effect was accompanied by increased levels of p53 and p21. This effect was abrogated in the presence of the protein kinase A (PKA) inhibitor Rp-8-bromoadenosine 3',5'-cyclic monophosphothioate. The increased levels of p53 and p21 observed in eNOS-transfected cells were reduced in the presence of the PKA inhibitor. These data suggest that p21 and p53 play a role in the inhibition of proliferation in eNOS-transfected cells and that levels of these two proteins are regulated by PKA.
cell proliferation; adenoviral gene transfer; nitric oxide; endothelial nitric oxide synthase; aorta
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INTRODUCTION |
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THE ENDOTHELIUM serves as the principal physiological source of nitric oxide (NO) in blood vessels (4, 5, 31). NO has been shown to have an inhibitory effect on vascular smooth muscle cell (VSMC) proliferation (12, 23). Endothelial damage and the disruption in NO production decrease the inhibitory effect of NO on VSMC proliferation. After endothelial injury, VSMCs undergo a "phenotypic modulation" and migrate from the media to the intima where they begin to proliferate and produce collagen (1). This results in vessel fibrosis, decreased cross-sectional area, and restenosis (4).
Endothelial nitric oxide synthase (eNOS) catalyzes the conversion of
L-arginine to L-citrulline and NO
(16). NO then diffuses from the endothelium into adjacent
VSMCs and binds to the heme moiety of soluble guanylate cyclase
(16). This binding results in activation of the enzyme and
an increase in smooth muscle cGMP concentration (14, 16,
24). Pig coronary arteries treated with the NO donor
S-nitroso-N-acetylpenicillamine (SNAP) showed increased cGMP levels, whereas cAMP levels were not altered
(21). Studies by Cornwell et al. (6) revealed
that in primary explants of aortic smooth muscle treated with either
IL-1 or NO donors, cGMP levels, but not cAMP levels, were increased.
This study also demonstrated that, in the presence of an NO donor, when
the activity of protein kinase A (PKA), but not that of protein kinase
G (PKG) was inhibited, VSMC proliferation in both treated and controls was similar (6). However, when PKG activity alone was
inhibited in NO donor-treated cells, proliferation was also inhibited.
It was, therefore, proposed that the increased cGMP levels in these cells were sufficient to activate PKA and that cross-activation of PKA
resulted in the inhibition of VSMC proliferation (6). However, the mechanism by which PKA acts to inhibit VSMC proliferation remains to be elucidated.
A potential downstream target for PKA is p21, also known as WAF1, CIP1, or SDI1. p21 itself is a downstream target for the p53 tumor suppressor gene (10). p53 has also been implicated in controlling cell passage (8, 9). In some cell types the induction of p21 has been shown to be p53 dependent (10, 28), whereas in other cell types this has been found not to depend on p53 (18, 28). The p21 gene has been reported to have a p53 transcriptional motif that enhances transcription of a number of genes, including p21 (27). p21 binds to cyclin/CDK complexes, blocking their activity, and results in the cell being unable to progress from the G1 to the S phase (15). Exogenous NO has been reported to affect the activity of p21 late in the G1 phase (18). In a study in VSMCs in vitro, p21 expression was increased by treatment with the NO donor SNAP (14). In another study, the p21 gene was transfected in vivo into porcine arteries injured by a balloon catheterization procedure and produced a 35% reduction in VSMC proliferation and a reduction in intimal hyperplasia (37). However, in platelet-derived growth factor (PDGF)-treated guinea pig coronary artery VSMCs transfected with the eNOS gene, a significant increase in p21 was not observed with the inhibition of VSMC proliferation (33). Thus the role of p21 in NO-induced inhibition of VSMC proliferation is uncertain. In the present study, we attempted to determine whether these cell cycle inhibitory proteins are involved in NO-induced inhibition of proliferation in eNOS-transfected aortic VSMCs. Recent studies of VSMC proliferation have focused on the transfer of specific genes, such as eNOS, p21, or retinoblastoma (Rb), in vivo or in vitro (3, 20, 30, 33). In the present study, the eNOS gene was transfected into rat aortic VSMCs in vitro, and the effect of eNOS transfection on cell proliferation was studied. It has been reported that PKA plays an important role in the inhibition of vascular VSMC proliferation following NO donor treatment (6). However, the PKA targets involved in the inhibition of VSMC proliferation have not yet been determined. The present study investigated the role of PKA in the inhibition of proliferation in eNOS-transfected cells and the involvement of the downstream effector molecules p53 and p21 in this process.
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METHODS |
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Cell culture and cell transfection.
Rat aortic VSMCs were obtained from male Sprague-Dawley rats weighing
~350-500 g. The rats were anesthetized with pentobarbital sodium
(35 mg/kg ip), and the aorta was removed and placed in tissue culture
medium 199 (Sigma Chemical, St. Louis, MO) (17). The
vessel was incubated in a collagenase solution (200 U/ml collagenase type I, 0.4 mg/ml trypsin inhibitor) for 30 min at 37°C. The
adventitia was removed, and the aorta was cut longitudinally. A sterile
cotton swab was used to disrupt the endothelial lining. The vessel was then minced into small pieces and placed in a collagenase-elastase solution (200 U/ml collagenase type I, 15 U/ml elastase, type III) for
2 h. The tissue was washed in tissue culture medium 199 supplemented with 10% FBS containing penicillin (100 U/ml) and streptomycin (200 µg/ml). The tissue pieces were plated on a
25-cm2 cell culture flask. The flask was placed in a
humidified incubator (95% air-5% CO2) at 37°C. The
tissue segments were allowed to attach for 5-7 days, after which
the medium in the flask was aspirated. Fresh medium supplemented with
FBS containing penicillin and streptomycin was added. Upon reaching
70% confluency, the cells were passaged by trypsinization (0.25%
trypsin, 0.053 mM EDTA; GIBCO, Grand Island, NY). The identity of the
cells was confirmed by the typical "hill-and-valley" appearance
exhibited by VSMCs. In addition, immunohistochemical studies showed
that >95% of the cells exposed to an NO donor stained positive for
smooth muscle-specific -actin.
Cell proliferation.
In all proliferation studies, 6 × 104 cells were
plated in each well of a six-well cell culture dish (Costar, Corning,
NY). The cells were transfected with 150, 300, or 450 multiplicity of
infection units (MOI) of AdCMVeNOS. Cell numbers were measured over the
course of 4 days. Once the optimal dose of the eNOS vector transfection
was determined in pilot studies, subsequent experiments were conducted
with 300 MOI AdCMVeNOS. Cell numbers in -galactosidase-transfected cells and nontransfected cells were compared to ensure that the adenoviral vector itself had no effect on proliferation.
Detection of eNOS.
After transfection, the presence of intracellular eNOS was
determined by using immunocytochemistry and Western blot analysis. For immunocytochemical detection, passage II cells were
plated on single-chambered slides (Nunc, Naperville, IL).
Nontransfected and cells transfected with AdCMVgal or AdCMVeNOS were
used for the immunohistochemical detection of eNOS. eNOS expression was determined on days 2 and 4 following
transfection. Immunohistochemistry was performed by using the
Immunocruz staining system (Santa Cruz Biotechnology, Santa Cruz, CA).
Medium was aspirated from the chamber slides, and the cells were fixed
in methanol (
10°C) for 5 min. To quench endogenous peroxidase
activity, we incubated the fixed cells in a 0.5% solution of hydrogen
peroxide in PBS for 7 min. Cells were washed in PBS and then incubated
in serum block for 20 min to prevent nonspecific binding. The cells
were immediately treated with the primary antibody eNOS (1:50)
(Transduction Laboratories, San Diego, CA) for 2 h. The cells were
washed in PBS and treated with biotinylated secondary antibody for 30 min, followed by treatment with horseradish peroxidase
(HRP)-streptavidin complex. The cells were then exposed to HRP solution
for 10 min. The cells were dehydrated by using 95% and 100% ethanol
and xylene. The slides were coverslipped and viewed under a light
microscope. The number of cells transfected was determined by counting
immunopositive cells.
Inhibition of eNOS activity with L-NAME.
The effect of the NOS inhibitor
N-nitro-L-arginine methyl ester
(L-NAME) on eNOS-transfected cells was investigated. The
effect of L-NAME on nontransfected cells was first
determined by treating passage II rat aortic VSMCs with
concentrations of 10
3, 10
4, and
10
5 M, and a dose-response study was then carried out in
eNOS-transfected cells. After it was determined that 10
4
M was an optimal dose in pilot studies, the cells were left untreated or were transfected with AdCMVeNOS or AdCMV
gal. In the
transfection experiments, the cells were preincubated with
L-NAME for 30 min before transfection, and cell counts were
obtained over the course of 4 days.
Effect of eNOS transfection and inhibition of PKA on expression
of p21 and p53.
Passage II rat aortic VSMCs were either left untreated or
transfected with -galactosidase or eNOS. One group of
eNOS-transfected cells was treated with Rp-8-BrcAMPS (100 µM). The cells were treated for 4 days and then harvested and lysed.
Protein levels were quantified, and equivalent amounts of protein (20 µg) for each group were run on a 4-20% gradient gel. Western
blot analysis was used to detect p21 and p53 levels. Monoclonal mouse
anti-p21 IgG (Santa Cruz Biotechnology) was used as the primary
antibody for the detection of p21, whereas monoclonal rabbit anti-p53
IgG (Santa Cruz Biotechnology) was used to detect p53. Bound antibody
was detected with anti-mouse IgG-HRP and anti-rabbit IgG-HRP secondary
antibodies. Bound antibody was visualized by using the ECL
chemiluminescence kit (Amersham).
Detection of apoptosis in eNOS-transfected cells. Studies were conducted to determine whether apoptosis was associated with increases in p21 and p53 levels in eNOS-transfected cells. The trypan blue exclusion test and Western blot analysis were used to access viability and the presence of apoptosis in cells infected with eNOS. Cells were exposed to AdCMVeNOS for 4 days, after which cell viability was determined. Cell viability was found to be >95% in rat aortic VSMCs transfected with AdCMVeNOS at 300 MOI. Cell proliferation was significantly inhibited at this dose of the adenoviral vector. For these studies, passage II rat aortic VSMCs were plated in six-well culture dishes. After attachment, the medium was aspirated and cells were either untreated or transfected with AdCMVeNOS at 300 MOI. Nontransfected cells were grown to either 95% confluency over a period of 4 days or harvested at 30% confluency. At 30% confluency, the cells were in log-phase growth. eNOS-transfected cells were incubated at 37°C for 4 days, after which the cells were trypsinized and lysed in hypotonic buffer. Protein samples were quantified by using the BCA protein assay (Pierce), and 20 µg of protein were loaded in each well in a 4-20% gradient gel. Western blot analysis was conducted by using anti-Bax IgG (Transduction Labs).
Statistical analysis. Cells were counted by using an Olympus IMT-2 microscope. Protein assays were carried out by using a Labsystems Multiskan MS plate reader (Labsystems, Franklin, MA). Western blot autoradiographs were scanned with an AlphaEase program (Alpha Innotech, San Leandro, CA). The data were analyzed statistically by using a one-way ANOVA, followed by post hoc analysis with Tukey's test.
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RESULTS |
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Proliferation studies in transfected cells.
As shown in Fig. 1A, after
treatment with 150 MOI AdCMVeNOS, cell proliferation was significantly
inhibited by day 4. Treatment with 300 MOI AdCMVeNOS also
significantly inhibited cell proliferation compared with nontransfected
cells. The inhibition of proliferation was maintained over the 4-day
period of the experiment. At 450 MOI AdCMVeNOS, cell proliferation was
significantly inhibited but cell numbers decreased by day 4 (Fig. 1A). In subsequent studies, the cells were treated
with 300 MOI AdCMVeNOS. Figure 1B shows cell counts carried
out in control and -galactosidase-transfected rat aortic VSMCs over
a 4-day period. There was no significant difference between the two
groups, and the doubling time for the AdCMV
gal-transfected cells was
the same as that observed in control cells.
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Detection of eNOS.
Immunohistochemical detection of eNOS was conducted on VSMCs
transfected with 300 MOI AdCMVeNOS. eNOS-transfected cells were compared with nontransfected and -galactosidase-transfected cells on
day 4 following transfer of the eNOS gene, and these results are shown in Fig. 2. Immunocytochemistry
was performed, and the cells transfected with AdCMVeNOS revealed the
presence of eNOS staining on day 4 (Fig. 2). Similar results
were obtained on days 1, 2, and 3 (data not shown). Transfection efficiency was between 80 and 90%, and
corresponding nontransfected and
-galactosidase-transfected cells
did not show the presence of eNOS staining.
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Inhibition of eNOS activity with L-NAME.
In eNOS-transfected cells, addition of L-NAME caused a
significant reduction of NO-induced inhibition of proliferation at concentrations of 103, 10
4, and
10
5 M (Fig. 4A).
Figure 4B shows the effect of L-NAME
(10
4 M) on eNOS- and
-galactosidase-transfected cells.
L-NAME did not appear to have an effect on
-galactosidase-transfected cells, whereas it prevented the
inhibition of proliferation in eNOS-transfected cells (Fig.
4B) .
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Effect of Rp-8-BrcAMPS and Rp-8-BrcGMPS on rat aortic VSMC
proliferation.
Figure 5A shows the effect of
three concentrations (25, 50, and 100 µM) of Rp-8-BrcAMPS
on proliferation in eNOS-transfected cells. A dose of 100 µM
Rp-8-BrcAMPS was found to block the inhibition of
proliferation (Fig. 5A). As shown in Fig. 5B, by
day 4 cell numbers in eNOS-transfected cells were
significantly lower than cell numbers in nontransfected and
-galactosidase-transfected cells. This finding suggests that
Rp-8-BrcAMPS blocked the PKA-mediated inhibitory effect of
eNOS-generated NO on VSMC proliferation (Fig. 5B).
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Expression of p21 and p53 in eNOS-transfected cells.
Figure 7A is representative of
a typical Western blot for p21 from three experiments, each run in
triplicate. Transfer of the eNOS gene to rat aortic VSMCs resulted in
an upregulation of p21. The levels of p21 were significantly higher in
eNOS-transfected cells compared with levels in nontransfected and
-galactosidase-transfected cells . In cells transfected with eNOS
and treated with Rp-8-BrcAMPS, p21 levels were found to be
significantly lower than in eNOS-transfected cells and not
significantly different from levels in nontransfected and in
-galactosidase-transfected cells (Fig. 7).
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Determination of apoptosis in eNOS-transfected cells. The trypan blue exclusion test on nontransfected cells (control) and eNOS-transfected cells showed that cell viability in both control and eNOS-transfected cells was >95%. There was no significant difference in cell viability between the two groups (data not shown). Additionally, the occurrence of apoptosis in eNOS-transfected cells was determined by using flow cytometry. Results of the flow cytometry experiments showed no increase in hypodiploidy in transfected cells compared with control cells (data not shown).
Figure 9A shows a representative Western blot for Bax, and Fig. 9B summarizes the mean data from Western blot analysis from three experiments, each carried out in triplicate. Levels of Bax in eNOS-transfected and nontransfected cells at 30% confluency were significantly lower than levels in nontransfected cells at 95% confluency, which are in a stationary phase (Fig. 9B). Nontransfected cells at 30% confluency showed levels of Bax similar to levels in eNOS-transfected cells. eNOS-transfected cells are typically inhibited at the 50% confluency level. Thus Bax levels in the eNOS-transfected cells (at 50% confluency) were equivalent to levels in nontransfected cells that were in log-phase growth (30% confluency). These results indicate that there was no significant increase in apoptosis in eNOS-transfected cells compared with levels in nontransfected control cells in log-phase growth.
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DISCUSSION |
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Results of the present study show that transfection of rats aortic
VSMCs with 300 MOI AdCMVeNOS significantly inhibited smooth muscle cell
proliferation. This effect was sustained through the course of the
experiment without affecting cell viability. At the 450 MOI dose of
AdCMVeNOS, cell numbers decreased below control level by day
4. These results suggest that doses of AdCMVeNOS higher than 300 MOI could be cytotoxic, and, therefore, the 300 MOI dose was used in
all subsequent experiments. The extent of eNOS transfection was
determined by immunocytochemical studies, and the presence of dark
brown staining confirmed the presence of the eNOS gene. Transfection
efficiency for this adenoviral vector was between 80 and 90%, and the
presence of eNOS was detected in cells as early as 1 day after
transfection. Western blot analysis and immunochemistry demonstrated
the presence of eNOS protein, and eNOS protein and staining were not
detected in nontransfected cells or in -galactosidase transfected
cells. To ensure that inhibition of proliferation occurred as a result
of NO production in eNOS-transfected cells, we investigated the effect
of the NOS inhibitor L-NAME. Inhibition of proliferation in
eNOS-transfected cells was reduced by L-NAME, suggesting
that eNOS overexpression increased the production of NO and providing
support for the hypothesis that the inhibition of VSMC proliferation by
eNOS gene transfer results from NO formation.
In addition to being associated with the inhibition of VSMC proliferation, NO has also been found to play a role in apoptosis. It has been reported that NO-induced upregulation of Fas was associated with increased apoptosis in rat and human VSMCs (11). However, in another study, NO was shown to inhibit Fas-induced apoptosis in human leukocytes (29). To ensure that transfer of the eNOS gene to rat aortic VSMCs was not cytotoxic, we evaluated cell viability using the trypan blue exclusion test and showed that at the dose (300 MOI) and transfection level observed, cell viability in nontransfected and eNOS-transfected cells was >95% on day 4. This finding indicated that at the concentration used in these studies, eNOS transfection was not cytotoxic to rat aortic VSMCs. In addition, Western blot analysis was performed to detect the apoptosis promoter Bax. Bax expression in eNOS-transfected cells was found to be equivalent to expression levels in proliferating, nontransfected cells (30% confluency) in log-phase growth. Bax levels in eNOS-transfected cells were lower than levels in nontransfected cells in the stationary phase (95% confluency).
In the present study, eNOS transfection resulted in growth arrest in
rat aortic VSMCs. Whereas adenoviral transfer of the eNOS gene was
found to have an inhibitory effect on proliferation, -galactosidase transfection had no effect on proliferation, and cell
numbers in AdCMV
gal-transfected cells were equivalent to numbers in
nontransfected cells up to 4 days after gene transfer. This finding
indicated that the inhibition of proliferation observed in eNOS
transfected cells was not dependent on the adenoviral vector but,
rather, on the introduction and biological activity of the eNOS gene itself.
A major advantage of adenoviral gene transfer is that the localization
of the gene can be achieved in organs, such as the lung, without having
systemic effects (2). Studies involving the transfer of
genes like p21 (37) and eNOS (36) suggest that gene transfection could serve as a possible approach to the treatment of intimal hyperplasia following angioplasty. eNOS
transfection has also been shown to have other potential therapeutic
uses. eNOS transfection to the lung of the mouse in vivo has been shown to reduce pulmonary vascular resistance. In that study, Champion et al.
(2) demonstrated that eNOS transfection was able to selectively reduce pulmonary pressor responses and to reduce the development of pulmonary hypertension. In another study, the eNOS gene
was transferred to the common carotid artery in rabbits
(30), and in a manner similar to that observed in our
study, Ooboshi et al. (30) were also able to detect eNOS
gene expression in transfected arteries by using immunohistochemical
techniques. In their study, in which arteries precontracted with
phenylephrine were used, eNOS-transfected vessels showed a greater
relaxation response to acetylcholine compared with responses in
nontransfected and -galactosidase-transfected arteries
(30). This finding suggests that eNOS transfection
enhanced the production of NO, which mediated the increased
vasorelaxant response (30). Thus eNOS transfection and NO
overproduction may be a useful technique for the study of NO-mediated
vascular responses.
In addition to studies involving p21 and eNOS gene transfection, the effects of Rb gene transfer have also been investigated (3). Cell proliferation was inhibited in rat aortic VSMCs transfected with the Rb gene in vitro (3). In that same study, in vivo arterial transfection with the Rb gene at the time of balloon angioplasty was found to significantly reduce neointimal thickening in rat carotid and porcine femoral arteries (3). While gene transfer of downstream effector molecules, such as p21 and Rb, could be useful in inhibiting VSMC proliferation associated with intimal hyperplasia, it is interesting to note that NO formed by eNOS has been shown to be effective in inhibiting VSMC proliferation and also for maintaining cells in a contractile phenotype (35, 36).
In determination of the role of p21 in PDGF-stimulated guinea pig coronary arteries, p21 protein levels did not increase in response to eNOS transfection (34). However, in the present study, an increase in p21 levels in eNOS-transfected rat aortic VSMCs was observed. The differential effects of eNOS on p21 expression may be attributed to the fact that the VSMCs used in the two studies were obtained from different sources. Another possible reason for this difference could be that PDGF was used to stimulate smooth muscle growth in the study of Sharma et al. (33), whereas this cytokine was not added in our studies. In contrast to the results of Sharma et al. (33), an increase in p21 levels, but not p53 expression, has been reported in studies using human umbilical VSMCs treated with the NO donor SNAP (18). In our studies, an induction of both p53 and p21 was observed in eNOS-transfected rat aortic VSMCs. An increase in p21 levels was also reported in a study by Sato et al. (32). In that study, eNOS gene transfer to VSMCs resulted in an upregulation of p21 and p27 levels. A similar upregulation in p21 was also reported in VSMCs exposed to SNAP or after inducible nitric oxide synthase (iNOS) gene transfer (26). In a more recent study, Ishida et al. (19) demonstrated that, contrary to results of their previous report, p53 was involved in the mediation of NO-induced expression of p21 in VSMCs. In the more recent publication, Ishida et al (19) attributed this discrepancy to the use of a different lysis buffer. The latter study was conducted with a stronger detergent, which resulted in more efficient extraction of p53 (19).
The study of Ishida et al. (19) reported that whereas p21 induction was dependent on p53 upregulation, it was not dependent on increases in cGMP concentration. The role of cGMP in NO-mediated inhibition of proliferation is uncertain. Cornwell et al. (6) proposed a cross-activation of PKA by cGMP. A study by Tanner et al. (34) indicated that the inhibitory effect of eNOS overexpression on human aortic VSMC proliferation was cGMP independent, whereas levels of cGMP were found to have increased in eNOS-transduced porcine coronary artery VSMCs (32). Kibbe et al. (25) determined that overexpression of iNOS inhibited VSMC proliferation in a cGMP-independent manner. It is possible that the involvement of cGMP may be dependent on the source of NO (NO donor as opposed to transfection with the NOS gene) or on the NOS isoform transfected into the cell. Thus, while the upstream regulators of PKA are still uncertain, our study indicates that PKA plays an important role in eNOS-induced inhibition of VSMC proliferation. In their study, Ishida et al. (19) also studied the effect of the PKA inhibitor Rp-8-BrcAMPS in SNAP-treated cells. They reported that at a concentration of 50 µM, the PKA inhibitor did not attenuate the effect of SNAP on [3H]TdR incorporation (19). In our study, the use of Rp-8-BrcAMPS at a concentration of 50 µM did not prevent the inhibition of proliferation in eNOS-transfected cells. However, our data show that when eNOS-transfected cells were exposed to a 100 µM concentration of Rp-8-BrcAMPS, the cells proliferated at the same rate as nontransfected cells. However, when eNOS-transfected cells were exposed to Rp-8-BrcGMPS, there was no effect on the inhibition of proliferation. These data suggest that PKA, but not PKG, was involved in NO-induced inhibition of proliferation. Whereas eNOS-transfected cells had increased p21 and p53 levels, eNOS-transfected cells in the presence of Rp-8-BrcAMPS showed a decrease in levels of these proteins. These data suggest that p21 and p53 were involved in NO-induced inhibition of proliferation and that the induction of these proteins was mediated by PKA. The induction of p21 and p53 was not found to be associated with apoptosis but, rather, with the inhibitory effect of NO on VSMC proliferation.
The present results suggest that PKA may be involved in the inhibition of proliferation in eNOS-transfected VSMCs by targeting the downstream effector molecules p53 and p21. At the transfection level attained in our studies, apoptosis did not appear to be a factor in the increased expression of p53 in eNOS-transfected cells. Whether PKA directly activates p53 and p21 or acts through another pathway remains to be determined, as do the upstream effector pathways for PKA. In conclusion, our results along with those from other studies suggest that eNOS gene transfer may have great potential for use in the treatment of cardiovascular diseases.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-62000, National Cancer Institute Grant CA-65600, and a grant from the American Heart Association Southeast Affiliate.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. R. Jeter, Jr., Dept. of Structural and Cellular Biology SL49, Tulane Univ. Health Sciences Center, 1430 Tulane Ave., New Orleans, LA 70112 (E-mail: jjeter{at}tulane.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published September 18;10.1152/ajpcell.00179.2002
Received 18 April 2002; accepted in final form 19 August 2002.
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