(Circulation. 2000;101:1982.)
© 2000 American Heart Association, Inc.
Clinical Investigation and Reports |
Nitric Oxide Modulates Expression of Cell Cycle Regulatory Proteins
A Cytostatic Strategy for Inhibition of Human Vascular Smooth Muscle Cell Proliferation
From the Cardiovascular Research (F.C.T., H.G., T.F.L.), Physiology Institute, University Zürich-Irchel; Division of Cardiology (F.C.T., T.F.L.), University Hospital, Zürich, Switzerland; Ophthalmology University Hospital (P.M., C.C.), Basel, Switzerland; and Division of Cardiology (E.G.N.), University of Michigan Medical Center, Ann Arbor, Mich.
Correspondence to Thomas F. Lüscher, MD, Cardiology, University Hospital, Rämistrasse 100, 8091 Zürich, Switzerland.
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Abstract |
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Background—We examined the effect of NO on the proliferation and cell cycle regulation of human aortic vascular smooth muscle cells (VSMCs).
Methods and Results—The NO donor diethylenetriamineNONOate (10-5 to 10-3 mol/L) inhibited proliferation in response to 10% fetal calf serum (FCS) and 100 ng/mL platelet-derived growth factor-BB in a concentration-dependent manner. This effect was not observed with disintegrated diethylenetriamineNONOate or with the parent compound, diethylenetriamine. Adenoviral transfection of endothelial NO synthase (NOS) inhibited proliferation in response to FCS, which was prevented with NG-nitro-L-arginine methyl ester. NOS overexpression did not inhibit proliferation in response to platelet-derived growth factor, although the transfection efficiency and protein expression were similar to those of FCS-stimulated cells. Nitrate release was selectively enhanced from FCS-treated cells, indicating that NOS was activated by FCS only. NO caused G1 cell cycle arrest. Cytotoxicity was determined with trypan blue exclusion, and apoptosis was assessed with DNA fragmentation. Cyclin-dependent kinase 2 expression level, threonine phosphorylation, and kinase activity were inhibited. Cyclin A expression was blunted, whereas cyclin E remained unchanged. p21 expression was induced, and p27 remained unaltered. The effect on cyclin A and p21 started within 6 hours and preceded the changes in cell cycle distribution. Proliferation in response to 10% FCS was barely inhibited with 8-bromo-cGMP (10-3 mol/L) but was blunted with both forskolin and 8-bromo-cAMP. Proliferation in response to 2% FCS was inhibited with 8-bromo-cGMP, but it did not mimic the cell cycle effects of NO.
Conclusions—NO inhibits VSMC proliferation by specifically changing the expression and activity of cell cycle regulatory proteins, which may occur independent of cGMP. Adenoviral overexpression of endothelial NOS represents a cytostatic strategy for gene therapy of vascular disease.
Key Words: endothelium-derived factors • nitric oxide • nitric oxide synthase • platelet-derived factors • gene therapy
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Nitric oxide (NO) inhibits platelet aggregation, induces vascular relaxation, and is involved in regulation of the proliferation of vascular smooth muscle cells (VSMCs).1 Indeed, NO donors inhibit VSMC proliferation,2 and the overexpression of NO synthase (NOS) in the rat carotid artery reduces neointima formation after balloon dilatation.3 4 5
Cell cycle progression is mediated by cyclin-dependent kinases (cdk).6 Progression in G1 phase and entry into S phase are related to the activity of cdk2 in complex with cyclin E and later with cyclin A.7 Cyclin E expression increases during G1 and peaks at G1-to-S transition; it enters into complexes with cdk2 throughout this time period.8 Cyclin A expression late in G1 is important for G1-to-S transition, because the inhibition of cyclin A kinase prevents S phase entry.9 The cdk activity is also affected by cyclin-dependent kinase inhibitors (cki).10 Both p21 and p27 are cki that interfere with cyclin E and cyclin A kinase activity. p21 is present in cdk complexes of proliferating cells during all phases of the cycle; the conversion of active into inactive complexes is achieved through alteration of the ratio of p21 to cdk.11 p27 levels are high in growth factor–deprived cells and decline in response to growth factor stimulation; mitogens are the main factor regulating the p27 level.12 Consistent with these observations in cultured cells, the vascular p27 level in vivo is inversely related to the degree of proliferation after balloon dilatation and thus permits proliferation in the presence of mitogens, whereas p21 is induced during the phase of declining proliferation and contributes to rendering the vessel quiescent.13
As NO inhibits the proliferation of VSMCs, changes in cell cycle regulation should occur under these conditions unless there was a cytotoxic or an apoptotic effect. The regulation of vascular tone by NO is related to the activation of soluble guanylate cyclase; however, the role of cGMP in the regulation of proliferation remains controversial.14 15 In the present study, we examined the effect of an NO donor and NOS transfection on the proliferation of VSMCs and analyzed the cell cycle regulation in the presence of NO and 8-bromo-cGMP.
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Methods |
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Recombinant Adenovirus
An adenoviral vector for the expression of human placental alkaline phosphatase (hpAP; AdhpAP) and a control virus without the transgene (Ad
E1) were prepared as described
previously.16 A similar virus for the expression of
endothelial NOS (eNOS; AdeNOS) and the respective
control virus were provided by Dr Stefan Janssens (Leuven, Belgium).
These viruses were derived from Ad5 dL309 and thus are slightly
different from Ad5 sub360.17 However, a comparison of the
effects of both control viruses on VSMC proliferation did not show any
difference (n=5; data not shown). Therefore, for the sake of clarity,
both control viruses are called AdE1. The titer of purified viruses
was determined with plaque assay on 293 cells with the use of an
adsorption time of 24 hours and with plaques counted on day 12 after
infection.18 Viral titers ranged from
2x1010 to 2x1011 pfu/mL
for all preparations. The titer of wild-type virus in the purified
preparations was determined with plaque assay on A549 cells under the
same conditions and was <1 in 109 pfu/mL for all
preparations.
Cell Culture and Cell Transfection
Human aortic VSMCs were obtained from Clonetics and maintained
in DMEM (GIBCO) containing 10% fetal calf serum (FCS; GIBCO). VSMCs
were used between passages 2 and 10. The 293 and A549 cells were
obtained from American Type Culture Collection and were cultured as
recommended. The transfection efficiency of VSMC was analyzed
with the use of AdhpAP and was determined 24 hours after infection as
described previously.19 An MOI (multiplicity of infection)
of 1000 pfu/cell was used for all experiments, because this MOI
resulted in the transfection of 99% of VSMCs under our experimental
conditions (n=4; data not shown). VSMCs infected with AdE1 and
noninfected cells served as negative controls for all experiments.
Proliferation and Nitrate Release
VSMCs were seeded at a density of 10 000 cells per 35-mm dish
and were cultured for 24 hours before drug treatment or adenovirus
infection. The cells were treated with diethylenetriamineNONOate
(DETANO) (10-5 to 10-3
mol/L; Alexis), diethylenetriamine (DETA) (10-3
mol/L; Sigma), spermineNONOate (10-5 to
10-3 mol/L; Alexis), spermine
(10-3 mol/L; Sigma), forskolin
(10-6 to 10-4 mol/L;
Calbiochem), 8-bromo-cGMP (10-5 to
10-3 mol/L; Sigma), 8-bromo-cAMP
(10-5 to 10-3 mol/L;
Sigma), and
NG-nitro-L-arginine
methyl ester (L-NAME; 4x10-3 mol/L; Sigma).
DETANO was chosen as the NO donor because it has a half-life of 27
hours and releases NO according to first-order kinetics. Moreover,
preliminary experiments demonstrated that spermineNONOate, but not
DETANO, induced a cytotoxic effect, which was related to the
spermine component of the drug (data not shown). The cells were
proliferating in a random manner in response to DMEM with 10% FCS when
DETANO treatment or adenoviral transfection was started. The cells were
maintained in DMEM containing 10% FCS, media were changed every day,
and fresh DETANO was added after every medium change. Cell number was
determined every other day for up to 6 days with the use of an
hematocytometer. To examine nitrate release, VSMCs were kept in medium
with either 10% FCS or 100 ng/mL platelet-derived growth factor-BB
(PDGF-BB) for 48 hours after transfection. Nitrate release was assessed
with the Griess reaction as described previously.20
Nitrate concentration was indicated in µmol/L and related to the
cell number.
Cell Cycle Distribution, Toxicity, and Apoptosis
VSMCs were seeded at a density of 250 000 cells per
150-mm dish and cultured for 24 hours before treatment with DETANO. The
cells were exposed to DETANO for 48 hours and were 
60% confluent at
the time of analysis. The cells were harvested and
analyzed for DNA content with the use of flow cytometry
(FACScan cytometer, CellQuest software; Becton Dickinson) as described
previously.21 For the examination of cytotoxicity, VSMCs
were gently trypsinized and evaluated for trypan blue exclusion. To
assess apoptosis, the percentage of propidium iodide–stained
cells with fragmented DNA was evaluated with the use of flow
cytometry.
Western Blot Analysis and H1 Kinase Assay
Western blot analysis was performed on whole-cell
lysates as described previously.22 Sixty micrograms of
protein was loaded per lane, resolved with SDS-PAGE under reducing
conditions, blotted onto PVDF membranes, and analyzed with the
use of chemiluminescence (Amersham). Equal loading of proteins was
controlled for through staining with Ponceau S and in selected
experiments with blotting for 
-tubulin as well. To determine cdk2
kinase activity, immunoprecipitations were performed as described
previously, the kinase reaction was performed for 30 minutes at 37°C
in the presence of 10 µCi of [
-32P]ATP
(Amersham) with 1 µg Histone H1 (Boehringer) as substrate,
and labeled proteins were resolved on SDS–15% polyacrylamide
gels, followed by autoradiography.22
Statistical Analysis
Results represent the mean value of 5 experiments as
indicated in the text. Data are expressed as mean±SEM, and statistical
comparisons were performed with Student’s t test for
unpaired observations or with ANOVA with Dunnett’s t test
correction whenever appropriate. A 2-tailed P value of
<0.05 was considered to indicate a statistically significant
difference.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Effect of NO on Proliferation
DETANO inhibited VSMC proliferation in response to 10% FCS (Figure 1A
) and to 100 ng/mL PDGF-BB
(Figure 1B) in a concentration-dependent manner. When DETANO was
used after having decayed during 4 half-lives, it barely inhibited
proliferation; similarly, the parent compound DETA did not affect cell
number (Figure 1C). DETANO had no toxic effect on VSMCs as
assessed with trypan blue (GIBCO) exclusion; after treatment for 2
days, the number of cells that excluded trypan blue was 9.77±0.38%
compared with 9.62±1.23% under control conditions (n=5; data no
shown).
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Adenoviral transfection of the eNOS cDNA inhibited VSMC proliferation
in response to 10% FCS (Figure 2B, left). This effect was dose dependent and resulted in a cell number of
87 750±5423 under control conditions, 74 938±5382 at an MOI of 100,
66 125±5524 at an MOI of 300, 62 063±3257 at an MOI of 1000, and
60 188±2339 at an MOI of 3000 compared with 42 250±1984 at the
beginning of the experiment (n=4; data not shown). However, no
inhibition was observed when proliferation was induced with 100 ng/mL
PDGF-BB (Figure 2B, right). Transfection efficiency was
identical because 99% of VSMCs stained for hpAP under each condition
(n=4; data not shown). The expression of eNOS protein also was
comparable (Figure 2A). In contrast, nitrate release was
enhanced in the presence of FCS (Figure 2C, left), whereas no
such effect was observed in the presence of PDGF (Figure 2C, right). The inhibition of proliferation after eNOS transfection was
prevented with the competitive inhibitor of eNOS, L-NAME
(Figure 3).
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Effect of NO on Cell Cycle Regulation
DETANO induced G1 phase arrest as
demonstrated with flow cytometric analysis (Figure 4A). There was only a slightly higher
number of cells with hypodiploid DNA (Figure 4A). The expression
of proteins that regulated G1 progression was
determined with Western blot analysis. In the presence of
DETANO, cyclin A expression was reduced within 24 hours, whereas cyclin
E was not affected. p21 level was transiently enhanced within 12 hours,
whereas p27 was not affected. cdk2 expression was reduced within 48
hours; this decline was moderate and protracted, because the protein
could still be detected after 48 hours. However, the
threonine-phosphorylated, and therefore active, cdk2,
represented by the lower band of the cdk2 signal, declined
faster than the total cdk2 and was no longer detectable within 48
hours. Consistent with these observations, cdk2 kinase activity
was blunted within 12 hours (Figure 4B). Similar to the
proliferation experiments, neither decayed DETANO nor the parent
compound DETA induced any change in the expression of cell cycle
proteins (data not shown).
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The time course of the changes in both cell cycle distribution and
protein expression was assessed every 6 hours over 48 hours to
correlate these alterations. The shift in cell cycle distribution
toward G1 started by 12 hours of exposure to
DETANO and progressively increased over 48 hours (Figure 5). The changes in expression of cyclin A
and p21 started by 6 hours and progressively increased over 48 hours,
indicating that these changes are primary events and responsible for
the G1 arrest (Figure 5). The expression
of both total cdk2 and threonine-phosphorylated cdk2
started to decline within 12 hours only, suggesting that the changes in
cdk2 level may occur secondary to the G1 arrest
(Figure 5).
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Effect of cGMP- or cAMP-Activating Substances
8-Bromo-cGMP (10-3 mol/L) barely affected
VSMC proliferation in response to 10% FCS, whereas DETANO completely
prevented proliferation at this concentration (Figure 6, left). In contrast, both 8-bromo-cAMP
and forskolin inhibited VSMC proliferation to a similar extent
(Table). 8-Bromo-cGMP (10-3
mol/L) inhibited proliferation in response to 2% FCS; the degree of
inhibition under these conditions was comparable to that with
10-4 mol/L DETANO (Figure 6, right). In
the presence of both 10% and 2% FCS, the expression of cell cycle
regulatory proteins was not affected by 10-3
mol/L 8-bromo-cGMP, whereas 10-3 and
10-4 mol/L DETANO, respectively, altered
expression in its typical manner (Figure 7).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In the present study, the NO donor DETANO inhibited VSMC proliferation in a concentration-dependent manner. Consistent with a specific effect, neither decayed DETANO nor the parent substance DETA affected cell number; furthermore, transfection of endothelial NOS inhibited proliferation as well. The effect of NO on proliferation was related to G1 arrest mediated by complete inhibition of cdk2 kinase activity due to a lower level of both cdk2 and cyclin A as well as by a higher level of p21 protein. 8-Bromo-cGMP was a very weak inhibitor of proliferation and did not induce any of the changes in the expression of cell cycle regulatory proteins observed with NO.
The normal proliferation rates in the presence of a competitive inhibitor of NOS, L-NAME, confirmed that the inhibition of proliferation after NOS transfection was mediated by NO. Although both transfection efficiency and protein expression were similar, this inhibition was observed only when proliferation was stimulated by FCS, not by PDGF-BB. Accordingly, nitrate release was only enhanced in the presence of FCS. Therefore, FCS appears to activate the recombinant enzyme, whereas this is not the case for PDGF. This interpretation is consistent with the observation that PDGF does not induce the release of NO from the endothelium, whereas many serum-derived products do.23
The cytostatic effect of NO was related to specific alterations in cell cycle regulation during G1 phase and is consistent with observations from other studies.24 25 26 27 Indeed, cdk2 kinase activity was blunted; this was in part due to a lower level of both total and threonine-phosphorylated cdk2.28 Because the decrease in cdk2 and the onset of G1 arrest occurred simultaneously, it is likely that the lower cdk2 level is a secondary event; indeed, cdk2 expression is maximal in S phase.28 Independent of its cellular level, cdk2 kinase activity is inhibited by cki. The level of p21 was transiently enhanced by NO. Because the overexpression of p21 induces G1 arrest in VSMCs, this elevated level is consistent with the antiproliferative effect of the nitrate.21 The importance of p21 is underscored by the observation that the increase in p21 preceded the onset of G1 arrest. In addition, the level of cyclin A was reduced in the presence of the nitrate. Because cdk2 exhibits kinase activity only when in a complex with a cyclin, the low level of cyclin A impaired cdk2 kinase activity as well. Similar to p21, the decrease in cyclin A preceded the onset of G1 arrest. Thus, it is conceivable that NO would induce G1 arrest in part of the cell population due to the higher level of p21; in addition, in cells that escape this block, NO would arrest proliferation in late G1 shortly before G1-to-S transition due to the lower level of cyclin A. The probably secondary decrease in both total and threonine-phosphorylated cdk2 would enhance the inhibitory effect on G1 progression. These observations support the concept that specific stimuli lead to distinct changes in G1 regulation. Indeed, some antiproliferative stimuli act on p21,29 others act on p27,30 and some act on cyclins.31 The pattern of alteration of cell cycle regulatory proteins in the presence of NO is unique and probably cell type specific.32
8-Bromo-cGMP was a much weaker inhibitor of proliferation than DETANO. However, the inhibitory effects of 8-bromo-cAMP and forskolin were comparable, indicating that bromide derivates of cyclic nucleotides can enter VSMCs in an effective manner. Consistent with the weak effect on proliferation, 8-bromo-cGMP did not affect the expression of any cell cycle regulatory protein that we examined. This was even the case when proliferation was stimulated with 2% FCS and the effect of 10-3 mol/L 8-bromo-cGMP on cell number was comparable to that of 10-4 mol/L DETANO. Thus, the effect of NO on cell cycle proteins does not seem to be mediated by cGMP. Because the expression pattern of these proteins after treatment with 8-bromo-cAMP is not compatible with that of NO, the effect of the nitrate also is not mediated by cAMP, although this was suggested in a previous study.15 The effect of NO may be mediated by other signal transduction pathways or be related to protein nitrosylation. Moreover, the effect of NO on p21, cdk2, and cyclin A may not be mediated by the same mechanism but rather may be regulated in an individual manner.
In summary, NO inhibits VSMC proliferation by inducing G1 phase arrest due to specific alterations in cell cycle regulation. The signal transduction processes that underlie this effect remain unknown, although neither cGMP nor cAMP seems to be involved. The cytostatic effect of NO suggests that the inhibition of neointima formation after balloon dilatation in the rat carotid artery is mediated not only by a reduced thrombus formation, leading to lower local concentrations of platelet-derived mitogens, but also by diminished VSMC proliferation due to a direct effect of NO on cell cycle progression.3 4 5 Therefore, other than cki such as p2121 or p2733 and the constitutively active retinoblastoma protein,34 the transfection of NOS represents a cytostatic gene transfer strategy and may have therapeutic potential for local treatment of vascular disease.
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Acknowledgments |
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This work was supported by the Swiss National Science Foundation (grants 31-47119.96 and 32-51069.97), the Swiss Heart Foundation, the Hartmann Müller Foundation, and the Schweizerische Rentenanstalt. The adenovirus for the expression of eNOS (AdeNOS) and the respective control virus were kindly provided by Dr Stefan Janssens (Leuven, Belgium).
Received April 16, 1999; revision received October 28, 1999; accepted November 15, 1999.
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I. L Williams, P. J Chowienczyk, S. B Wheatcroft, A. G Patel, R. A Sherwood, A. M Shah, and M. T Kearney Divergent effects of angiotensin-converting enzyme inhibition on blood pressure and endothelial function in obese humans Diabetes and Vascular Disease Research, May 1, 2006; 3(1): 34 - 38. [Abstract] [PDF]
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D. Kong, L. G. Melo, A. A. Mangi, L. Zhang, M. Lopez-Ilasaca, M. A. Perrella, C. C. Liew, R. E. Pratt, and V. J. Dzau Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells Circulation, April 13, 2004; 109(14): 1769 - 1775. [Abstract] [Full Text] [PDF]
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R.P. Mason, P. Marche, and T.H. Hintze Novel Vascular Biology of Third-Generation L-Type Calcium Channel Antagonists: Ancillary Actions of Amlodipine Arterioscler Thromb Vasc Biol, December 1, 2003; 23(12): 2155 - 2163. [Abstract] [Full Text] [PDF]
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P. Viani, P. Giussani, L. Brioschi, R. Bassi, V. Anelli, G. Tettamanti, and L. Riboni Ceramide in Nitric Oxide Inhibition of Glioma Cell Growth. EVIDENCE FOR THE INVOLVEMENT OF CERAMIDE TRAFFIC J. Biol. Chem., March 7, 2003; 278(11): 9592 - 9601. [Abstract] [Full Text] [PDF]
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F. M. D'Souza, R. L. Sparks, H. Chen, P. J. Kadowitz, and J. R. Jeter Jr. Mechanism of eNOS gene transfer inhibition of vascular smooth muscle cell proliferation Am J Physiol Cell Physiol, January 1, 2003; 284(1): C191 - C199. [Abstract] [Full Text] [PDF]
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Y. Nakamura, Y. Yamagata, N. Sugino, H. Takayama, and H. Kato Nitric Oxide Inhibits Oocyte Meiotic Maturation Biol Reprod, November 1, 2002; 67(5): 1588 - 1592. [Abstract] [Full Text] [PDF]
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M. T Gewaltig and G. Kojda Vasoprotection by nitric oxide: mechanisms and therapeutic potential Cardiovasc Res, August 1, 2002; 55(2): 250 - 260. [Abstract] [Full Text] [PDF]
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L. M.A.B. Van Bortel, H. A.J. Struijker-Boudier, and M. E. Safar Pulse Pressure, Arterial Stiffness, and Drug Treatment of Hypertension Hypertension, October 1, 2001; 38(4): 914 - 921. [Abstract] [Full Text] [PDF]
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J. L. Tuttle, R. D. Nachreiner, A. S. Bhuller, K. W. Condict, B. A. Connors, B. P. Herring, M. C. Dalsing, and J. L. Unthank Shear level influences resistance artery remodeling: wall dimensions, cell density, and eNOS expression Am J Physiol Heart Circ Physiol, September 1, 2001; 281(3): H1380 - H1389. [Abstract] [Full Text] [PDF]
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