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(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

Felix C. Tanner, MD; Peter Meier, MD; Helen Greutert, BS; Claudine Champion, BS; Elizabeth G. Nabel, MD; Thomas F. Lüscher, MD

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.

	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 N^G-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

	Introduction

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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.

	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 (AdE1) 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 N^G-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.

	Results

Top Abstract Introduction Methods Results Discussion References

 
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).

View larger version (24K): [in this window] [in a new window]   Figure 1. A, Effect of NO donor DETANO on proliferation of human aortic VSMCs in response to 10% FCS. FCS increased cell number within 6 days from 10 115±513 to 76 700±4759. DETANO reduced this effect to 71 200±5234 at 10-5 mol/L, 39 700±3270 at 10-4 mol/L, and 5900±623 at 10-3 mol/L. B, Effect of DETANO on proliferation of VSMCs in response to 10% FCS or 100 ng/mL PDGF-BB. FCS increased cell number within 2 days from 18 050±1025 to 32 100±924. In presence of DETANO, cell number was 18 100±1337. PDGF increased the cell number within 2 days from 12 950±950 to 21 400±1410. In presence of DETANO, cell number was 13 200±1340. C, Effect of fresh compared with decayed DETANO on proliferation of VSMCs in response to 10% FCS. FCS increased cell number within 2 days from 18 450±1723 to 39 400±1231. Fresh DETANO reduced this effect to 21 750±651, whereas cell number remained at 35 200±1604 with decayed DETANO and at 39 200±892 with parent compound DETA.

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).

View larger version (22K): [in this window] [in a new window]   Figure 2. A, Expression of eNOS in human aortic VSMCs after adenoviral transfection: Western blot analysis shows presence of eNOS protein in cells infected with eNOS adenovirus (AdeNOS) but not with control adenovirus (AdE1) or in uninfected cells. This expression pattern was identical in cells stimulated with 10% FCS and 100 ng/mL PDGF-BB. B, Effect of adenoviral transfection of eNOS cDNA on proliferation of VSMCs in response to 10% FCS or 100 ng/mL PDGF-BB. FCS increased cell number within 2 days from 31 780±3345 to 64 625±3565. In adenovirus-treated cells, cell number was 65 500±4500 with AdE1 and 33 125±2894 with AdeNOS (P<0.0001 vs control and vs AdE1). PDGF increased cell number within 2 days from 26 353±2928 to 45 900±2329. In adenovirus-treated cells, cell number was 44 775±2734 with AdE1 and 43 875±3097 with AdeNOS (P=NS vs control and vs AdE1). C, Effect of adenoviral transfection of eNOS cDNA on nitrate release from VSMCs in response to 10% FCS or 100 ng/mL PDGF-BB. In FCS-stimulated cells, nitrate release was 16.50±0.73 with AdeNOS and 8.25±0.50 with AdE1 compared with 9.49±0.16 µmol/Lx10 000/cell number/well in noninfected VSMCs (P<0.0001 for AdeNOS vs AdE1 or vs control, P=NS for AdE1 vs control). In PDGF-stimulated cells, nitrate release was 9.94±0.37 with AdeNOS and 7.13±0.59 with AdE1 compared with 9.58±0.35 µmol/Lx10 000/cell number/well in noninfected VSMCs (P=NS for AdeNOS vs control).

View larger version (32K): [in this window] [in a new window]   Figure 3. Effect of L-NAME on inhibition of human aortic VSMC proliferation induced by adenoviral transfection of eNOS. Cell number increased within 2 days from 16 295±1568 to 36 300±2035. In adenovirus-treated cells, cell number was 28 875±437 with control adenovirus (AdE1) and 20 550±515 with eNOS adenovirus (AdeNOS; P<0.0001 vs control and P=0.0005 vs E1). In L-NAME–treated cells, cell number increased to 32 525±1178 without adenovirus, 25 425±1035 with AdE1, and 24 775±669 with AdeNOS (P<0.0001 vs control and P=NS vs AdE1).

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).

View larger version (25K): [in this window] [in a new window]   Figure 4. A, Effect of NO donor DETANO (10-3 mol/L) on cell cycle distribution of human aortic VSMCs. Under control conditions, 64.0±0.84% of cells were in G0/G1 phase, 17.4±0.68% were in S phase, and 18.6±0.81% were in G2/M phase. After treatment with DETANO (10-3 mol/L) for 2 days, 81.8±0.86% of cells were in G0/G1 phase, 5.4±0.51% were in S phase, and 12.8±0.49% were in G2/M phase. Under control conditions, sub-G1 peak was slightly lower (1.60±0.29%) compared with that with DETANO treatment (7.80±1.36%). B, Effect of DETANO on expression of cell cycle regulatory proteins in VSMCs. Cyclin A started to decline within 12 hours of treatment with DETANO (10-3 mol/L) and was no longer detectable within 24 hours. In contrast, cyclin E was not affected. p21 was transiently induced within 12 hours of treatment with DETANO. In contrast, p27 was not affected. cdk2 started to decline within 12 hours of treatment with DETANO. This decline was moderate and protracted; protein could still be detected after 48 hours of treatment with DETANO. However, threonine-phosphorylated, and therefore active, cdk2, represented by lower band of cdk2 signal, declined faster and was no longer detectable within 48 hours. As a result of all of these changes, cdk2 kinase activity was blunted within 12 hours of treatment with DETANO.

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).

View larger version (38K): [in this window] [in a new window]   Figure 5. Time course of effect of NO donor DETANO (10-3 mol/L) on changes in cell cycle distribution and expression of cell cycle regulatory proteins in human aortic VSMCs. Within 12 hours of exposure to DETANO, cell cycle distribution started to shift toward G1 phase and progressively increased over 48 hours. Expression of cyclin A was inhibited and p21 was induced; these changes were first observed within 6 hours of exposure to DETANO. Expression of cdk2 was moderately reduced so that it could still be detected after 48 hours. However, the threonine-phosphorylated, and therefore active, cdk2, represented by the bottom band of cdk2 signal, declined faster and was no longer detectable within 48 hours. Expression of both total and threonine-phosphorylated cdk2 started to decrease within 12 hours of exposure to DETANO. Expression of both cyclin E and p27 remained unaltered during a 48-hour observation period; expression of -tubulin as a control for equal protein loading remained unaltered as well.

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).

View larger version (15K): [in this window] [in a new window]   Figure 6. Effect of NO donor DETANO compared with 8-bromo-cGMP on proliferation of human aortic VSMCs in response to 10% or 2% FCS. Ten percent FCS increased cell number within 4 days from 9805±650 to 46 350±3172. DETANO (10-3 mol/L) reduced this number to 6550±431, whereas it remained at 34 650±1786 in presence of 8-bromo-cGMP (10-3 mol/L). Two percent FCS increased cell number within 4 days from 11 012±1189 to 18 562±3968. DETANO (10-4 mol/L) reduced this number to 13 662±2908, and 8-bromo-cGMP (10-3 mol/L) reduced it to 11 449±3560.

View this table: [in this window] [in a new window]   Table 1. Effect of Forskolin and 8-Bromo-cAMP on Proliferation of Human Aortic VSMCs

View larger version (57K): [in this window] [in a new window]   Figure 7. Effect of NO donor DETANO compared with 8-bromo (br)-cGMP on expression of cell cycle regulatory proteins in human aortic VSMCs. Effect of DETANO was assessed at 10-3 mol/L, when proliferation was stimulated with 10% FCS, and at 10-4 mol/L, when it was stimulated with 2% FCS. Cyclin A declined to an undetectable level, whereas cyclin E was not affected. p21 was transiently induced, whereas p27 was not affected. cdk2 declined to a lower level. In contrast, 10-3 mol/L 8-bromo-cGMP did not affect expression of any cell cycle regulatory protein with either 10% or 2% FCS.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
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.

	Acknowledgments

 
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.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. 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;83:1774–1777.
   
2. Mooradian DL, Hutsell TC, Keefer LK. Nitric oxide (NO) donor molecules: effect of NO release rate on vascular smooth muscle cell proliferation in vitro. J Cardiovasc Pharmacol. 1995;25:674–678.[Medline] [Order article via Infotrieve]
   
3. Van der Leyen HE, Gibbons GH, Morishita R, Lewis NP, Zhang L, Nakajima M, Kaneda Y, 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.[Abstract/Free Full Text]
   
4. Janssens S, Flaherty D, Nong ZX, Varenne O, Vanpelt N, Haustermans C, Zoldhelyi P, Gerard R, Collen D. Human endothelial cell nitric oxide synthase gene transfer inhibits vascular smooth muscle cell proliferation and neointima formation after balloon injury in rats. Circulation. 1998;97:1274–1281.[Abstract/Free Full Text]
   
5. Chen L, Daum G, Forough R, Clowes M, Walter U, Clowes AW. Overexpression of human endothelial nitric oxide synthase in rat vascular smooth muscle cells and in balloon-injured carotid arteries. Circ Res. 1998;82:862–870.[Abstract/Free Full Text]
   
6. Sherr CJ. Cancer cell cycles. Science. 1996;274:1672–1677.[Abstract/Free Full Text]
   
7. Sherr CJ. G1 phase progression: cycling on cue. Cell. 1994;79:551–555.[Medline] [Order article via Infotrieve]
   
8. Hinds PW, Mittnacht S, Dulic V, Arnold A, Reed SI, Weinberg RA. Regulation of retinoblastoma protein functions by ectopic expression of human cyclins. Cell. 1992;70:993–1006,.[Medline] [Order article via Infotrieve]
   
9. Pagano M, Pepperkok R, Verde F, Ansorge W, Draetta G. Cyclin A is required at two points in the human cell cycle. EMBO J. 1992;11:961–971.[Medline] [Order article via Infotrieve]
   
10. Sherr CJ, Roberts JM. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev. 1995;9:1149–1163.[Free Full Text]
    
11. Zhang H, Hannon GJ, Beach D. p21-containing cyclin kinases exist in both active and inactive states. Genes Dev. 1994;8:1750–1758.[Abstract/Free Full Text]
    
12. Coats S, Flanagan WM, Nourse J, Roberts JM. Requirement of p27^Kip1 for restriction point control of the fibroblast cell cycle. Science. 1996;272:877–880.[Abstract]
    
13. Tanner FC, Yang Z, Duckers E, Gordon D, Nabel GJ, Nabel EG. Expression of cyclin-dependent kinase inhibitors in vascular disease. Circ Res. 1998;82:396–403.[Abstract/Free Full Text]
    
14. Yu SM, Hung LM, Lin CC. cGMP-elevating agents suppress proliferation of vascular smooth muscle cells by inhibiting the activation of epidermal growth factor signaling pathway. Circulation. 1997;95:1269–1277.[Abstract/Free Full Text]
    
15. Cornwell TL, Arnold E, Boerth NJ, Lincoln TM. Inhibition of smooth muscle cell growth by nitric oxide and activation of cAMP-dependent protein kinase by cGMP. Am J Physiol. 1994;C1405–C1413.
    
16. Ohno T, Gordon D, San H, Pompili VJ, Imperial MJ, Nabel GJ, Nabel EG. Gene therapy for vascular smooth muscle cell proliferation after arterial injury. Science. 1994;265:781–784.[Abstract/Free Full Text]
    
17. Alcorn JL, Gao E, Chen Q, Smith ME, Gerard RD, Mendelson CR. Genomic elements involved in transcriptional regulation of the rabbit surfactant protein-A gene. Mol Endocr. 1993;7:1072–1085.[Abstract]
    
18. Mittereder N, March KL, Trapnell BC. Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy. J Virol. 1996;70:7498–7509.[Abstract]
    
19. Tanner FC, Carr DP, Nabel GJ, Nabel EG. Transfection of human endothelial cells. Cardiovasc Res. 1997;35:522–528.[Abstract/Free Full Text]
    
20. Schmidt HHW, Kelm M. Determination of nitrite and nitrate by the Griess reaction. In: Feelisch M, Stamler JS, eds. Methods in Nitric Oxide Research. Chichester, NY: Wiley; 1996:491–497.
    
21. Yang Z, Simari RD, Perkins ND, San H, Gordon D, Nabel GJ, Nabel EG. Role of the p21 cyclin-dependent kinase inhibitor in limiting intimal cell proliferation in response to arterial injury. Proc Natl Acad Sci U S A. 1996;93:7905–7910.[Abstract/Free Full Text]
    
22. Matsushime H, Quelle DE, Shurtleff SA, Shibuya M, Sherr CJ, Kato JY. D-type cyclin-dependent kinase activity in mammalian cells. Mol Cell Biol. 1994;14:2066–2076.[Abstract/Free Full Text]
    
23. Tanner FC, Noll G, Boulanger CM, Lüscher TF. Oxidized low density lipoproteins inhibit relaxations of porcine coronary arteries: role of scavenger receptor and endothelium-derived nitric oxide. Circulation. 1991;83:2012–2020.[Abstract/Free Full Text]
    
24. Sarkar R, Gordon D, Stanley JC, Webb RC. Cell cycle effects of nitric oxide on vascular smooth muscle cells. Am J Physiol. 1997;272:H1810–H1818.[Abstract/Free Full Text]
    
25. Ishida A, Sasaguri T, Kosaka C, Nojima H, Ogata J. Induction of the cyclin-dependent kinase inhibitor p21^{Sdi1/Cip1/Waf1} by nitric oxide-generating vasodilator in vascular smooth muscle cells. J Biol Chem. 1997;272:10050–10057.[Abstract/Free Full Text]
    
26. Guo K, Andres V, Walsh K. Nitric oxide-induced downregulation of Cdk2 activity and cyclin A gene transcription in vascular smooth muscle cells. Circulation. 1998;97:2066–2072.[Abstract/Free Full Text]
    
27. Sharma RV, Tan E, Fang S, Gurjar MV, Bhalla RC. NOS gene transfer inhibits expression of cell cycle regulatory molecules in vascular smooth muscle cells. Am J Physiol. 1999;276:H1450–H1459.[Abstract/Free Full Text]
    
28. Gu Y, Rosenblatt J, Morgan DO. Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15. EMBO J. 1992;11:3995–4005.[Medline] [Order article via Infotrieve]
    
29. Dulic V, Kaufmann WK, Wilson SJ, Tlsty TD, Lees E, Harper JW, Elledge SJ, Reed SI. p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest. Cell. 1994;76:1013–1023.[Medline] [Order article via Infotrieve]
    
30. Nourse J, Firpo E, Flanagan WM, Coats S, Polyak K, Lee MH, Massague J, Crabtree G, Roberts J. Interleukin-2-mediated elimination of p27^Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin. Nature. 1994;372:570–573.[Medline] [Order article via Infotrieve]
    
31. Fang F, Orend G, Watanabe N, Hunter T, Ruoslahti E. Dependence of cyclin E-cdk2 kinase activity on cell anchorage. Science. 1996;271:499–502.[Abstract]
    
32. Takagi K, Isobe Y, Yasukawa K, Okouchi E, Suketa Y. Nitric oxide blocks the cell cycle of mouse macrophage-like cells in the early G2+M phase. FEBS Lett. 1994;340:159–162.[Medline] [Order article via Infotrieve]
    
33. Chen D, Krasinski K, Sylvester A, Chen J, Nisen PD, Andres V. Downregulation of cyclin-dependent kinase 2 activity and cyclin A promoter activity in vascular smooth muscle cells by p27(KIP1), an inhibitor of neointima formation in the rat carotid artery. J Clin Invest. 1997;99:2334–2341.[Medline] [Order article via Infotrieve]
    
34. Chang MW, Barr E, Seltzer J, Jiang YQ, Nabel GJ, Nabel EG, Parmacek MS, Leiden JM. Cytostatic gene therapy for vascular proliferative disorders with a constitutively active form of the retinoblastoma gene product. Science. 1995;267:518–522.[Abstract/Free Full Text]
    

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