© 2000 American Heart Association, Inc.
Cellular Biology |
Loss of Expression of the ß Subunit of Soluble Guanylyl Cyclase Prevents Nitric Oxide–Mediated Inhibition of DNA Synthesis in Smooth Muscle Cells of Old Rats
From the Departments of Surgery (L.C., G.D., J.W.F., S.H., A.W.C.), University of Washington, Seattle, and the Department of Pathology (M.-L.B.-P., G.G.), University of Geneva, CMU, Geneva, Switzerland.
Correspondence to Dr Lihua Chen, Departments of Surgery, Box 356410, University of Washington, Seattle, WA 98195. E-mail lihua{at}u.washington.edu
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Abstract—We compared the effects of NO donors and cGMP analogues on the growth of aortic smooth muscle cells (SMCs) derived from newborn, adult (aged 3 months), and old (aged 2 years) rats. We found that the NO donor S-nitroso-N-acetylpenicillamine failed to block DNA synthesis in SMCs from old rats but was effective in SMCs from newborn and adult rats. However, cGMP analogues were inhibitory in all 3 SMC types. We demonstrated that in SMCs from old rats, NO was unable to increase the concentration of intracellular cGMP, suggesting that either cGMP synthesis was defective or cGMP degradation was enhanced. Western blot analysis revealed that SMCs from old rats do not express the ß subunit of soluble guanylyl cyclase. To confirm the importance of this observation in vivo, we balloon-injured the carotid arteries of adult and old rats. Whereas soluble guanylyl cyclase was expressed at the same level in the media of injured vessels and uninjured vessels of both groups, its expression in the intimas of old rats was reduced by 70% compared with intimas from adult animals. Furthermore, N
-nitro-L-arginine, an
inhibitor of NO synthesis, enhanced the intimal thickening
in injured vessels in adult rats but not in old rats. We conclude that
the loss of NO responsiveness in aged rats is due to the lack of the
ß subunit of soluble guanylyl cyclase, and we speculate that this
defect contributes to the enhanced intimal thickening in response to
injury in old animals.
Key Words: cGMP • hyperplasia • nitric oxide • smooth muscle cells • soluble guanylyl cyclase
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Introduction |
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The principal function of NO in large arteries is to regulate vascular tone. This effect is mediated by the activation of the heme-containing soluble guanylyl cyclase (sGC) and the subsequent increase of cGMP levels.1 2 3 4 5 NO might also be an inhibitor of smooth muscle cell (SMC) proliferation and migration. NO donors or NO generated by the overexpression of NO synthase inhibits DNA synthesis and the growth of cultured SMCs.6 7 8 Oral delivery of NO donors or L-arginine (a NO precursor) and local transfer of endothelial constitutive NO synthase inhibit intimal formation after balloon injury.9 10 11 12 13 14 These results demonstrate that NO can blunt the injury response in vivo. Most studies have suggested that sGC and cGMP are the major mediators for the antiproliferative effects of NO on SMCs.2 15 16 One of the major downstream events after an increase in intracellular cGMP is the activation of cGMP-dependent protein kinase (PKG).15 16 It is reasonable to think that the NO-mediated inhibitory effect on SMC growth might be through PKG. However, the loss of PKG expression in passaged SMCs seems to cause the cells to adopt a "synthetic phenotype" even though the cells still respond to NO donors.17 18 19 20 This finding provides support for the possibility that NO can inhibit SMC growth by a pathway that does not depend on PKG. These results do not eliminate the possibility of involvement of cGMP in NO-mediated inhibition of SMC proliferation.
Aging might affect the ability of SMCs to proliferate in response to vascular injury.21 Several observations indicate that the response of SMCs to injury is greater in older rats compared with younger rats. SMC proliferation and intimal thickening in balloon-injured vessels are enhanced in older rats.22 23 24 Likewise, cultured aortic SMCs from old rats exhibit a more dedifferentiated phenotype and grow more rapidly in response to serum and platelet-derived growth factor than do SMCs from younger animals.25 26 Vessels from young and old rats have been cross-transplanted into syngeneic hosts of different ages; only the vessels from old rats exhibit a marked intimal thickness in response to injury that is independent of the age of the host.21 This finding indicates that the enhanced neointimal response is determined by the age of the vessel donor.
To investigate whether age could modify the response of SMCs to NO, we compared the effects of NO donors and cGMP analogues on the growth of aortic SMCs derived from newborn, adult (aged 3 months), and old (aged 2 years) Fischer rats. We investigated the downstream events in NO signaling, including intracellular cGMP levels and activation of cGMP-dependent protein kinase (PKG) in these 3 types of SMCs. We also examined the expression of the ß subunit of sGC (sGCß) in these 3 types of SMCs in vitro and in rat balloon-injured carotid arteries in vivo.
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Materials and Methods |
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Materials
Rabbit polyclonal anti–vasodilator-stimulated phosphoprotein (anti-VASP), anti–PKG Iß sera, and mouse monoclonal anti–VASP phosphoserine 239 antibody were generous gifts from Drs Ulrich Walter and Suzanne M. Lohmann (University of Würzburg, Würzburg, Germany). The rabbit anti-sGC serum was purchased from Alexis Co. Other chemicals were purchased from Sigma Chemical Co.
Cell Cultures
Adult SMC cultures were prepared by enzymatic digestion of
aortas from 3-month old Fischer 344 rats (Simonsen Laboratories,
Gilroy, Calif) as described.8 SMCs from newborn (aged <5
days) and old (aged 2 years) Fischer rats were prepared as
described.26 The cells were propagated in DMEM containing
10% FBS (GIBCO Laboratories) in 5% CO2 at
37°C7 and were used between passages 6 and 15.
DNA Synthesis
DNA synthesis was measured as described8 by use of
[3H]thymidine incorporation in response to
stimulation of 10% FBS for 24 hours in the presence or absence of
appropriate reagents
(S-nitroso-N-acetylpenicillamine [SNAP] or cGMP
analogues). The passage numbers of SMCs from animals of different ages
were the same in each experiment.
Measurement of Intracellular cGMP Levels
The cells were seeded at 4x105 cells per
well in 6-well tissue culture plates. The cells were extracted, and the
intracellular cGMP concentration was determined by a cGMP enzyme
immunoassay system according to the manufacturer’s protocol (Amersham
Co).
Protein Extraction and Western Blotting
Cultured SMCs and frozen tissues were extracted in HEB buffer
(25 mmol/L HEPES [pH 7.5], 5 mmol/L EDTA, 5 mmol/L
EGTA, 150 mmol/L NaCl, 100 mmol/L
Na4P2O7,
50 mmol/L NaF, 1 mmol/L benzamidine, 1% Triton X-100, 10%
glycerol, 0.1% ß-mercaptoethanol, 1 µg/mL pepstatin-A, 5 µg/mL
leupeptin, and 5 µg/mL aprotinin).27 The same amount of
protein was heated for 5 minutes at 95°C, then subjected to 10%
SDS-PAGE, and transferred to nitrocellulose membranes (Bio-Rad). The
blots were submerged in 5% milk in TTBS (25 mmol/L Tris-HCl,
500 mmol/L NaCl, and 0.1% Triton X-100), followed by an
incubation at 4°C overnight in 0.1% milk in TTBS containing the
appropriate primary antibody in concentrations recommended by the
manufacturer or references. The proteins were detected by horseradish
peroxidase–labeled secondary antibody with the use of a standard
enhanced chemiluminescence protocol provided by the manufacturer
(Amersham Co). Relative protein quantification was performed by
scanning autoradiographs.
Animal Experiments and Tissue Preparation
Male Fischer 344 rats, aged 3 months (220 to 330 g) from
Simonsen Laboratories (Gilroy, Calif) or aged 24 months (370 to
500 g) from the National Institutes of Health (NIH,
Bethesda, Md), were anesthetized, and the left carotid
arteries were surgically exposed. The distal half of the common carotid
arteries was isolated, and the endothelium was stripped
by the passage of a 2F balloon catheter (V. Mueller) introduced through
an arteriotomy in the external branch. The external carotid arteries
were then ligated after removal of the catheter, the blood flow was
restored, and the wound was closed. Animals were fed with either
H2O or H2O plus
N-nitro-L-arginine
(L-NA), an inhibitor of NO synthesis (10 mg/kg per day),
for 2 weeks. Systolic arterial blood pressures were
measured in conscious restrained rats by tail-cuff plethysmography
(Norco Biosystems). After 2 weeks, the animals were euthanized, and the
arteries were flushed clear of blood with Ringer’s lactate solution.
Arteries intended for Western blotting analysis were excised.
The neointima and media in injured carotid arteries were
separated under a microscope, immediately frozen in liquid nitrogen,
and stored at -70°C. Arteries intended for
histological analysis were flushed clear of
blood with Ringer’s lactate solution and fixed by perfusion with 10%
neutral buffered formalin, pH 7.4, at 100 mm Hg and embedded in
paraffin for histology.8 Measurement of luminal,
intimal, and medial areas was made on 2 cross sections per carotid
taken from the middle of each carotid segment. All surgical procedures
were performed according to the Principles of Laboratory Animal
Care and the Guide for the Care and Use of Laboratory
Animals (NIH publication No. 86-23, revised 1985).
Statistics
All values are expressed as mean±SD. Comparisons among the
groups were made by the Mann-Whitney nonparametric test.
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Results |
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NO Donor SNAP Fails to Inhibit DNA Synthesis and to Increase cGMP Levels in Old SMCs
To investigate the influence of age on NO-mediated growth inhibition, we compared the effect of the NO donor SNAP and cGMP analogues 8-bromo-cGMP and dibutyryl-cGMP on DNA synthesis in rat aortic SMCs derived from newborn, adult (aged 3 months), and old (aged 2 years) animals. DNA synthesis in response to 10% FBS was inhibited by both cGMP analogues in all 3 groups by 40% (Figure 1A
). The NO donor SNAP, however, had no
effect on SMCs from old rats but decreased DNA synthesis in SMCs from
newborn and adult rats by 25% (Figure 1B). This observation
supported the conclusion that NO failed to increase cGMP levels in SMCs
from old rats.
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To confirm this conclusion, we measured the induction of cGMP on SNAP
treatment. In SMCs from newborn and adult rats, SNAP increased the cGMP
concentration 25- and 10-fold, respectively, but it failed to elevate
cGMP levels in SMCs from old rats (Figure 2).
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cGMP-Dependent Protein Kinase Is Expressed and Functional in All 3
SMC Groups
Because it has been suggested that cultured SMCs may lose
expression of the cGMP-dependent protein kinase
(PKG),17 18 19 20 we tested whether PKG was functional and
could account for the inhibitory effect of cGMP in all 3
rat SMC groups. To monitor the PKG activity in cells, we used the shift
assay with the PKG substrate VASP. VASP is a 46-kDa protein of yet
unknown function that has been identified on the basis of its
phosphorylation by PKG.28 29 The
phosphorylated form of VASP migrates in
SDS-polyacrylamide gels as a 50-kDa protein.29 On
treatment of SMCs with 8-bromo-cGMP, a 50-kDa band of VASP was detected
by Western blotting in all 3 SMC types (Figure 3A). Consistent with our
observation that NO does not increase cGMP levels in SMCs from old
rats, NO induced VASP phosphorylation in SMCs from
newborn and adult rats only (Figure 3A). VASP was
phosphorylated in SMCs from newborn and adult rats on
SNAP treatment in a dose-dependent manner (Figure 3B). SNAP
failed to induce VASP phosphorylation in SMCs from old
rats even at the highest concentration (250 µmol/L, Figure 3A) and over a long period of time (up to 24 hours, data not
shown). To confirm that cGMP analogue and SNAP-induced cGMP
activate PKG, which, in turn, phosphorylates VASP,
we used a Western blotting assay with the monoclonal antibody against
VASP phosphoserine 239. This site is specifically
phosphorylated by PKG30 but not protein
kinase A (PKA), even though PKA is also capable of phosphorylating
VASP.28 29 The results indicate that VASP is
phosphorylated by PKG in response to 8-bromo-cGMP in
all 3 cell types and in response to SNAP in SMCs from newborn and adult
rats (Figure 4A). VASP
phosphorylation induced by a cAMP analogue,
dibutyryl-cAMP, a PKA activator, could not be detected by
the antibody to phosphoserine 239 (Figure 4A), even though VASP
phosphorylation in response to cAMP analogues could be
detected with an antibody to the other 2 PKA-dependent VASP
phosphorylation sites.29 Furthermore, the
presence of PKG protein in all 3 SMC types could be demonstrated by
Western blotting (Figure 4B). These results indicate that PKG
was expressed and functional in all 3 types of SMCs, but NO was not
able to activate PKG in SMCs from old rats.
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Old SMCs Do Not Express sGCß
The fact that NO failed to induce cGMP production in SMCs
from old rats suggests either that sGC is not functional or that cGMP
is rapidly degraded. sGC is a heterodimer consisting of 
and ß
subunits, both of which are catalytically active and targeted by
NO.31 When we measured the expression levels of sGCß by
Western blotting, we detected the protein in SMCs from newborn and
adult rats but not in SMCs from old rats (Figure 5A). To rule out the possibility that
SMCs from old rats do express a NO-responsive sGC that we failed to
detect, we measured cyclic nucleotide levels on SNAP
treatment in the absence and presence of 3-isobutyl-1-methylxanthine
(IBMX), a general cyclic nucleotide phosphodiesterase
inhibitor. The addition of IBMX increased cAMP but not cGMP
levels whether or not SNAP was present (Figure 5B). This
observation is consistent with the hypothesis that NO fails to
produce cGMP in SMCs from old rats because they lack a functional sGC.
The loss of guanylyl cyclase activity in SMCs from old rats seems to be
specific for the heme-containing sGC. The atrial
natriuretic peptide that binds and activates the
type A membrane guanylyl cyclase was able to increase the intracellular
cGMP level by 5-fold (from 2.8±0.8 to 14.0±2.0 fmol/µg, n=3) in
SMCs from old rats as well as in SMCs from newborn and adult
rats.32 As expected, we also observed atrial
natriuretic peptide–induced VASP
phosphorylation in all 3 cell types (data not
shown).
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In Old Rats, Intimal SMCs Exhibited Decreased Expression of sGCß
After Balloon Injury
We investigated the possibility that an age-dependent loss of sGC
expression plays a role in the response to injury in rat carotid
arteries. The left carotid arteries of adult and old rats were
balloon-injured and harvested after 2 weeks. The media and intima of
the injured carotid were separated, and the proteins were extracted and
analyzed by Western blotting for the expression of sGC. As a
control, the noninjured right carotid artery was processed in the same
way. Both adult and old rats expressed similar levels of sGCß in the
uninjured carotid arteries. After injury, however, intimal SMCs from
old but not adult animals exhibited an average 70% decrease in sGC
levels (Figure 6). No differences between
adult and old rats were observed in the media of the injured carotid
arteries.
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In Old Rats, Intimal Formation After Balloon Injury Was Not
Affected by L-NA, an Inhibitor of NO Synthesis
To establish the relevance of decreased sGC expression in
intimal formation in old rats, we investigated the effect of L-NA
feeding on intimal formation after balloon injury in old and adult
rats. Blood pressures were significantly increased in both age groups
receiving L-NA (at 2 weeks, old rats: control 115±8.0 mm Hg,
n=8; L-NA 150±6.3 mm Hg, n=11; adult rats: control 122±8.2
mm Hg, n=11; L-NA 157±9.3 mm Hg, n=11). The ratio of intima to
media in old rats was not significantly changed by L-NA (0.95±0.17
[control, n=8] versus 0.92±0.22 [L-NA, n=11], P=0.75),
whereas this ratio was significantly increased in adult rats (0.61±0.18 [control, n =11] versus 0.82±0.23 [L-NA, n=11],
P=0. 03). This result suggests that NO release in adult rat
arteries inhibited intimal thickening, whereas it had no effect in old
rat arteries. A likely explanation for this result is that intimal SMCs
of adult rats express sGCß and, therefore, are responsive to NO,
whereas the old rat SMCs lacking sGCß cannot respond to NO and,
therefore, are unaffected by L-NA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NO is a vasodilator and a negative regulator of SMC function. On the basis of the observation that in the rat carotid artery subjected to balloon injury, older Fischer rats develop a bigger neointima than do younger rats,21 we investigated whether the SMC response to NO is lost as the animals grow older. The proliferation of SMCs derived from newborn and adult SMCs, but not SMCs from old rats, was inhibited by NO (Figure 1B
). In
contrast, all 3 SMC types were inhibited when the cGMP analogue was
used (Figure 1A). It has previously been reported that cultured
SMCs exhibit reduced expression of the cGMP-dependent protein
kinase.17 18 19 20 Our data, however, support the conclusion
that the defect in the SMCs from old Fischer rats lies upstream from
PKG. We measured the expression levels and activity of PKG and found
that the kinase was indeed present and functional in the SMCs from
old rats (Figures 3 and 4). The element in the NO pathway
missing in the SMCs from old rats was found to be sGC, the direct
intracellular target of NO. As determined by Western blotting, the ß
subunit of the enzyme was detectable only in SMCs from newborn and
adult rats (Figure 5). It is noteworthy that the loss of
guanylyl cyclase was specific for sGC in SMCs from old rats, in view of
the fact that the guanylyl cyclase associated with the atrial
natriuretic peptide receptor was present and
functional. The failure of NO to increase cGMP was not attributable to increased cGMP degradation, in view of the fact that cGMP levels did not change in the absence and presence of the general phosphodiesterase inhibitor, IBMX.
Does the loss of sGCß with aging affect the injury response? There
was a 70% decrease in sGCß in the intima of old rats (Figure 6) even though the expression of sGC in the normal vessels and
in the intima of adult rats was the same (Figure 6). Part of the
response of SMCs to vessel injury is the upregulation of inducible
NOS.33 It is possible that medial SMCs produce NO that not
only dilates the injured vessel but also limits the extent of
neointimal hyperplasia. Intimal formation was augmented by
L-NA in adult rat arteries but was not affected in old rat arteries.
These results support the conclusion that the lack of sGCß in intimal
SMCs renders these cells unresponsive to locally synthesized NO.
Furthermore, the observation that intimal thickening in adult rats is
increased by L-NA treatment confirms the importance of NO in limiting
the extent of intimal thickening after injury. It is not clear why
intimal SMCs in the old animals do not have a functional sGC inasmuch
as they are derived from the media in which the enzyme is apparently
present. Phenotypic heterogeneity and clonality has
been reported for SMCs in the rat vessel wall.34 35
Because medial SMCs are permanently exposed to
endothelium-derived NO, it is possible that aging
favors the appearance of SMC clones lacking a functional sGC. Those
clones may be the ones that migrate and proliferate after vessel
injury. Another possibility is that the old SMCs are losing sGC in the
course of forming the neointima. Future experiments will be
designed to distinguish between these 2 possibilities and to delineate
the molecular mechanism of how the expression of sGCß is switched
off.
In summary, we present a novel mechanism to explain how SMCs become insensitive to the growth-inhibitory effects of NO. It is an interesting possibility that a loss of NO inhibition caused by a loss of a functional sGC may also occur in humans and contribute to the higher risk in elderly people of developing restenosis after angioplasty.
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Acknowledgments |
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This study was supported by NIH grant HL-52459. We thank Drs Suzanne M. Lohmann and Ulrich Walter for anti-PKG, anti-VASP, and anti–VASP phosphoserine 239 antibodies and Drs Joseph A. Beavo, Karin Bornfeldt, and George Martin for helpful discussions. We thank Holly Lea and Patti Polinsky for technical assistance with the in vivo experiments.
Received October 28, 1999; accepted December 1, 1999.
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A. Friebe and D. Koesling Regulation of Nitric Oxide-Sensitive Guanylyl Cyclase Circ. Res., July 25, 2003; 93(2): 96 - 105. [Abstract] [Full Text] [PDF]
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P. Sinnaeve, J.-D. Chiche, H. Gillijns, N. Van Pelt, D. Wirthlin, F. Van de Werf, D. Collen, K. D. Bloch, and S. Janssens Overexpression of a Constitutively Active Protein Kinase G Mutant Reduces Neointima Formation and In-Stent Restenosis Circulation, June 18, 2002; 105(24): 2911 - 2916. [Abstract] [Full Text] [PDF]
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P. Sinnaeve, J.-D. Chiche, Z. Nong, O. Varenne, N. Van Pelt, H. Gillijns, D. Collen, K. D. Bloch, and S. Janssens Soluble Guanylate Cyclase {{alpha}}1 and {beta}1 Gene Transfer Increases NO Responsiveness and Reduces Neointima Formation After Balloon Injury in Rats via Antiproliferative and Antimigratory Effects Circ. Res., January 19, 2001; 88(1): 103 - 109. [Abstract] [Full Text] [PDF]
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