Arteriosclerosis, Thrombosis, and Vascular Biology. 1998;18:1771-1779
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1998;18:1771-1779.)
© 1998 American Heart Association, Inc.
Natriuretic Factors and Nitric Oxide Suppress Plasminogen Activator Inhibitor-1 Expression in Vascular Smooth Muscle Cells
Role of cGMP in the Regulation of the Plasminogen System
Julie L. Bouchie;
Hans Hansen;
;
Edward P. Feener
From the Research Division, Joslin Diabetes Center, Harvard Medical
School, Boston, Mass.
Correspondence to Edward P. Feener, PhD, Research Division, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. E-mail feenere{at}joslab.harvard.edu
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Abstract
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AbstractIncreased expression of
plasminogen activator
inhibitor-1
(PAI-1) has been reported in atherosclerotic and
balloon-injured
vessels. Little is known regarding the factors
and mechanisms that may
negatively regulate PAI-1 expression.
In this report, the effect of
cGMP-coupled vasoactive hormones,
including natriuretic
factors and nitric oxide, on the regulation
of PAI-1 expression in
vascular smooth muscle cells was examined.
Atrial
natriuretic factor 128 (ANF) and C-type
natriuretic
factor-22 (CNP) reduced angiotensin
II (Ang II) and platelet-derived
growth factorstimulated PAI-1
mRNA expression in rat aortic
smooth muscle cells by 50% to 70%, with
corresponding reductions
in PAI-1 protein release. Treatment of human
aortic smooth muscle
cells with CNP similarly inhibited both
platelet-derived growth
factorinduced PAI-1 mRNA expression and
PAI-1 protein
release by 50%. Dose-response studies revealed that the
inhibitory
effects of CNP and ANF on PAI-1 expression were
concentration
dependent, with IC
50s of

1 nmol/L for both
natriuretic peptides.
Ang IIstimulated PAI-1 expression
was also inhibited by
the nitric oxide donor
S-nitroso-
N-acetylpenicillamine. The
membrane-permeant
cGMP analogue 8-Br-cGMP reduced Ang IIstimulated
PAI-1
expression by 60%, and an inhibitor of soluble
guanylyl cyclase
(1
H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one)
significantly
impaired the inhibitory effects of
S-nitroso-
N-acetylpenicillamine
on Ang
IIstimulated PAI-1 expression. Studies of PAI-1
mRNA stability in
cells treated with actinomycin D showed that
ANF did not alter PAI-1
mRNA half-life, suggesting that natriuretic
factors reduce
PAI-1 transcription. These data show that natriuretic
factors
and nitric oxide, via a cGMP-dependent mechanism, inhibit PAI-1
synthesis
in vascular smooth muscle cells. Thus, cGMP-coupled
vasoactive
hormones may play an important role in suppressing vascular
PAI-1
expression.
Key Words: natriuretic factors nitric oxide plasminogen activator inhibitor vascular smooth muscle cells angiotensin II
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Introduction
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The plasminogen system plays an integral role
in the regulation
of vascular physiology by regulating
fibrinolysis and the proteolysis
of extracellular
matrix (ECM).
1 2 Plasminogen
activator inhibitor-1
(PAI-1), the principle
inhibitor of the plasminogen system,
irreversibly
inactivates both tissue and urokinase
plasminogen activators.
3
Inhibition of plasminogen activation by PAI-1 impairs
fibrinolysis
and thereby promotes
thrombosis.
4 5 In addition, PAI-1 has
been shown
to regulate ECM turnover and vascular smooth muscle
cell (VSMC)
migration,
6 7 2 key processes in vascular
remodeling
and atherogenesis.
Circulating PAI-1 levels are increased in a variety of
pathophysiological conditions, including insulin
resistance, obesity, noninsulin-dependent diabetes mellitus (NIDDM),
and insulin-dependent diabetes mellitus with
microalbuminuria.8 9 10
Cross-sectional studies have identified elevated plasma PAI-1 as a risk
factor for myocardial infarction in several high-risk
populations.11 12 13 In addition, increased levels
of PAI-1 also occur within the vascular wall at sites of
atherosclerotic lesions,14 15 16
neointimal formation of failing bypass vein
grafts,17 and balloon-injured
vessels.18 19 Upregulation of PAI-1 within
diseased or injured vascular tissue may locally diminish
plasminogen activation and contribute to the accumulation
of fibrin and ECM within vascular lesions. Conversely, a local increase
in plasminogen activation potential within the vascular
wall, associated with a relative increase in tissue
plasminogen activator and urokinase
plasminogen activator expression compared with
that of PAI-1, has been shown at sites of abdominal aortic
aneurysms.20 21 Within these sites, high
levels of plasmin may locally increase proteolysis of ECM and thereby
physically weaken the vascular wall.
A number of growth factors, hormones, and cytokines, including
platelet-derived growth factor (PDGF), angiotensin II
(Ang II), transforming growth factor-ß, and tumor necrosis
factor-
, induce PAI-1 expression in
VSMCs.22 23 24 25 26 However, although the processes that
upregulate PAI-1 expression have received considerable attention, much
less is known regarding the factors and mechanisms that may negatively
regulate PAI-1 expression in vascular cells. In this report, we have
examined the effect of 2 types of vasorelaxant compounds,
natriuretic factors and nitric oxide, on the regulation of
PAI-1 expression in VSMCs. In addition to regulating vascular tone,
atrial natriuretic factor (ANF), C-type
natriuretic peptide (CNP), and nitric oxidegenerating
compounds inhibit VSMC growth and migration in
vitro27 28 29 30 and reduce neointimal
formation after balloon catheterinduced vascular
injury.31 32 Nitric oxide has also been shown to
provide antithrombotic activity33 34 35 and reduce
PAI-1 released from platelets.36 37 As such,
exogenous local delivery of nitric oxide synthase and CNP has been
proposed as a treatment to reduce
restenosis.31 38 However, little is known
regarding the roles of natriuretic factors and nitric oxide
in the regulation of the plasminogen system. In this
report, we show that ANF, CNP, and a nitric oxide donor, acting via a
cGMP-dependent process, are potent inhibitors of PAI-1
expression in VSMCs. These data suggest that natriuretic
factors and nitric oxide may represent a class of cGMP-coupled
vasorelaxant hormones that suppress vascular PAI-1 expression.
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Methods
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Cell Culture
Rat aortic smooth muscle cells (RASMCs) were isolated from
Sprague-Dawley
rats, cultured in Dulbecco's modified Eagle's medium
(DMEM),
100 mg/dL
D-glucose (Gibco-BRL), and 10% FBS
(Gibco BRL) as
described previously
23 and used
between passages 8 and 15.
Human aortic smooth muscle cells (HASMCs)
were isolated from
aortic medial explants as described in Reference 39
39 ,
cultured
in DMEM with 20% FBS, and used between passages 7 and 10.
Confluent
monolayers of cells were deprived of serum in DMEM containing
0.1%
(wt/vol) BSA for 18 hours before stimulation. Cells were
stimulated
with Ang II (Sigma Chemical Co) or PDGF (Upstate Biotech) in
the
absence or presence of a 5-minute pretreatment with atrial
natriuretic
factor 128 (ANF), C-type
natriuretic peptide-22 (CNP,
Peninsula), or
S-nitroso-
N-acetylpenicillamine (SNAP, Sigma)
unless
indicated otherwise. The role of cGMP in the regulation of PAI-1
was
determined in cells treated with 8-bromo-cGMP (Calbiochem),
1
H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one
(ODQ), and
KT5823 (Biomol).
RNA Isolation and Northern Blot Analysis
Total RNA was isolated by using TRI reagent (Molecular Research
Center). Fifteen micrograms of RNA was separated in a 1% (wt/vol)
agarose gel containing 20 mmol/L MOPS, pH 7.0; 5 mmol/L
sodium acetate; 1 mmol/L EDTA; 0.76 µg/mL ethidium bromide; and
0.67% formaldehyde. The RNA was transferred to Biotrans membranes (ICN
Pharmaceuticals) and cross-linked by UV. A cDNA probe against rat
PAI-123 was labeled by using the Multiprime DNA
labeling system (Amersham Corp) and purified with a NICK column
(Pharmacia LKB Biotechnology Inc). A human PAI-1 antisense
oligonucleotide (Oncogene Science Inc) was end-labeled
by using T4 polynucleotide kinase and
[32P]ATP and then purified with a NAP-5 column
(Pharmacia LKB). Blots were hybridized in 50 mmol/L PIPES,
100 mmol/L NaCl, 50 mmol/L NaPO4,
1 mmol/L EDTA, 0.1% salmon sperm DNA (Biostar), and 5% SDS at
65°C. The blots were then washed with 0.5x SSC and 5% SDS at
65°C. Levels of mRNA were visualized and quantified by PhosphorImage
analysis (Molecular Dynamics Inc). RNA loading was normalized
to acidic ribosomal phosphoprotein PO (36B4) by using a
[32P]ATP end-labeled
oligonucleotide probe.
Western Blot Analysis
PAI-1 protein levels were measured as described
previously.23 Conditioned media from RASMCs or
HASMCs, treated with Ang II and PDGF in the absence or presence of ANF
and CNP, was collected. Equal aliquots of conditioned media were
separated by 10% SDSpolyacrylamide gel electrophoresis and
transferred to nitrocellulose (Novex). Western blotting was performed
with anti-rat PAI-1 antibody and anti-human PAI-1 antibody (American
Diagnostica, Inc) followed by detection with enhanced
chemiluminescence (ECL, Amersham). PAI-1 was visualized and quantified
using PhosphorImage analysis and ImageQuant software (Molecular
Dynamics). PAI-1 levels were normalized to total protein from cell
lysates by using the Bradford method (Bio-Rad).
cGMP Immunoassay
Confluent 6-well dishes of serum-deprived RASMCs were stimulated
with 100 nmol/L ANF, CNP, or 10 µmol/L SNAP in the presence of
0.5 mmol/L 1-methyl-3-isobutylxanthine for 15 minutes. Cells were
then washed with ice-cold PBS containing 1-methyl-3-isobutylxanthine
and lysed in HCl. Levels of cGMP were measured by a cGMP enzyme
immunoassay kit (Biomol).
Statistics
All statistical analyses were performed by 1-way ANOVA
with SigmaStat software (Jandel Scientific). Values of
P<0.05 were considered significantly different.
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Results
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Effect of Natriuretic Peptides on PAI-1 mRNA Expression
in RASMCs and HASMCs
The effects of both ANF and CNP on the regulation of PAI-1
expression
in RASMCs and HASMCs were examined both alone and in the
presence
of the strong stimulators of PAI-1 expression, Ang II and
PDGF.
22 23 24 Control cells and cells
pretreated with natriuretic factors
for 5 minutes
were stimulated with either Ang II or PDGF for
3 hours. Northern blot
analyses of both PAI-1 and acidic ribosomal
phosphoprotein PO
mRNA (36B4, a constitutively expressed mRNA)
were performed as
described previously.
23 A dose response of
Ang
II's and PDGF's effects on PAI-1 mRNA levels in RASMCs revealed
a
maximal induction of PAI-1 levels by

30- and 40-fold, respectively
(Figure
1

, left and right), confirming
that these agonists are potent
stimulators of PAI-1 expression. The
effect of ANF on PAI-1
expression in RASMCs was examined in cells
pretreated with 100
nmol/L ANF for 5 minutes, followed by a 3-hour
stimulation with
Ang II and PDGF. This study showed that ANF inhibited
the maximal
levels of Ang II and PDGFinduced PAI-1 expression
by
70% and 50%, respectively (Figure 1

, left and right). In
addition,
ANF appeared to cause a small rightward shift in the
concentration
dependence of PDGF-stimulated PAI-1 that was not
apparent in Ang
IItreated cells. The dose-response effect
of ANF on Ang
IIstimulated PAI-1 expression showed that
ANF decreased PAI-1 levels
in a concentration-dependent manner,
with its maximal
inhibitory effect obtained at 10 nmol/L and
an
IC
50 of <3 nmol/L (Figure 2

, top left).

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Figure 1. Effect of ANF on Ang II and PDGF-stimulated
PAI-1 mRNA expression in RASMCs. Cells were stimulated with the
indicated concentrations of Ang II (left) or PDGF (right) for 3 hours
in the absence (circles) or presence (triangles) of a 5-minute
pretreatment with 100 nmol/L ANF. Total RNA was harvested from cells,
and PAI-1 and 36B4 mRNA levels were determined by Northern blot
analysis. Results were visualized and quantified by
PhosphorImage analysis, and representative
Northern blots of PAI-1 and 36B4 mRNA are shown. Graphs
represent PAI-1 mRNA levels normalized to 36B4 mRNA from 3
independent experiments for each condition.
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Figure 2. Dose response and time course of ANF's
and CNP's effects on Ang IIstimulated PAI-1 expression in RASMCs.
Cells were pretreated for 5 minutes with the indicated concentrations
of ANF (top left) or CNP (top right) followed by the addition of 100
nmol/L Ang II for 3 hours. Representative Northern
blots and graph quantification of PAI-1 mRNA normalized to 36B4 from 3
independent experiments are shown. Data are presented as
percent of PAI-1 mRNA levels in the presence of Ang II alone. Controls
(Con) indicate PAI-1 expression in the absence of Ang II or
natriuretic peptides. Significant differences
(P<0.01, ANOVA) of PAI-1 levels from cells treated with
natriuretic factors versus Ang II alone are indicated as
**. Bottom left, The effect of CNP when administered before (-30 and
-5 minutes) and after (+30 minutes) the introduction of Ang II (100
nmol/L, 3 hours). Northern blots of PAI-1 and 36B4 are shown. Bar graph
represents PAI-1 mRNA levels normalized to 36B4 from 2
experiments performed in triplicate.
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To determine whether this effect was specific for ANF or whether it
could also occur with other natriuretic peptides, the
effect of CNP on PAI-1 expression in RASMCs was examined. Treatment of
cells with CNP inhibited Ang IIstimulated PAI-1 expression in a
concentration-dependent manner, with its maximal inhibition of 60%
occurring at 10 nmol/L and an IC50 of
1 nmol/L
(Figure 2
, top right). These studies demonstrated that ANF and CNP
exhibited similar maximal inhibitory effects and dose
dependence on PAI-1 expression in RASMCs. A time course of CNP's
inhibitory effect on Ang IIstimulated PAI-1 expression
was also examined. PAI-1 mRNA expression was compared in RASMCs treated
with 100 nmol/L CNP at 30 minutes before, 5 minutes before, or 30
minutes after the start of Ang II stimulation (100 nmol/L, 3 hours).
CNP reduced PAI-1 expression by 59% to 67% over this time interval
(Figure 2
, bottom left). These results revealed that pretreatment of
cells with CNP was not required to inhibit Ang IIstimulated PAI-1
expression.
To determine whether natriuretic peptide could also
regulate PAI-1 expression in HASMCs, the effect of CNP on PAI-1
expression in HASMCs was examined both alone and under PDGF-stimulated
conditions. Northern blot analysis of human PAI-1 mRNA revealed
2 transcripts of 2.3 and 3.2 kb, as described
previously.14 Stimulation of cells with PDGF (25
ng/mL) increased the level of both PAI-1 transcripts by 2- to 3-fold
(Figure 3
). Treatment of cells with 100
nmol/L CNP significantly inhibited PDGF-stimulated PAI-1 expression by
50% for the 3.2-kb transcript (P<0.01, ANOVA) and
inhibited the 2.3-kb PAI-1 transcript to a similar extent. To our
knowledge, this is the first description of a factor that can inhibit
PAI-1 expression in human vascular cells.

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Figure 3. Effect of CNP on PDGF-stimulated PAI-1 mRNA in
HASMCs. HASMCs were stimulated with PDGF (25 ng/mL) for 3 hours in the
absence or presence of a 5-minute pretreatment with CNP (100 nmol/L).
PAI-1 mRNA levels were determined by Northern blot analysis,
and representative blots showing 3.2- and 2.3-kb PAI-1
mRNA transcripts and 36B4 are shown. Bar graph represents
3.2-kb PAI-1 mRNA levels normalized to 36B4. Significant difference
(P<0.01, ANOVA) is indicated with **. Con indicates
control.
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Effect of Natriuretic Peptides on the Release of PAI-1
Protein From RASMCs and HASMCs
To determine whether the inhibitory effects of
natriuretic peptides on PAI-1 mRNA resulted in changes in
PAI-1 synthesis at the protein level, the effect of ANF and CNP on
PAI-1 protein released from RASMCs and HASMCs was examined. RASMCs were
pretreated with ANF (100 nmol/L) or CNP (100 nmol/L) for 5 minutes
followed by the addition of Ang II (100 nmol/L). After an 18-hour
incubation, PAI-1 protein levels in the conditioned media were measured
by Western blot analysis. PAI-1 immunoblotting
revealed a band at 52 kDa, the expected size of
PAI-1.5 Treatment of cells with 100 nmol/L Ang II
increased the level of PAI-1 protein in the conditioned medium by
5-fold (Figure 4
, left). Addition of
either ANF or CNP significantly (P<0.05, ANOVA) decreased
PAI-1 protein released from these cells by 80% and 60%, respectively,
compared with cells stimulated with Ang II alone (Figure 4
, left). The
inhibitory effects of ANF and CNP on PAI-1 protein levels
were not significantly different. In a similar series of experiments
with HASMCs, the effects of CNP on PAI-1 protein levels from control
and PDGF-stimulated cells were examined. Stimulation of HASMCs with
PDGF increased PAI-1 protein in the condition medium by
2-fold
(Figure 4
, right). Treatment of cells with 100 nmol/L CNP did not alter
basal released PAI-1 levels but reduced PDGF-stimulated PAI-1 antigen
by 50% (P<0.05, ANOVA). Thus, the inhibitory
effects of ANF and CNP on PAI-1 mRNA expression in rat and human SMCs,
described above, correspond with quantitatively similar decreases in
PAI-1 protein released from these cells to the conditioned media.

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Figure 4. Effect of ANF and CNP on PAI-1 protein released
from RASMCs and HASMCs. Left, RASMCs were pretreated with 100 nmol/L
ANF or CNP for 5 minutes followed by the addition of Ang II (100
nmol/L) for 18 hours. Right, HASMCs were treated with CNP (100 nmol/L)
for 5 minutes followed by an 18-hour incubation with PDGF (25 ng/mL).
PAI-1 protein in the culture medium was determined by Western blot
analysis using anti-rat PAI-1 and anti-human PAI-1 antibodies
for left and right panels, respectively. Results were visualized by
ECL, and representative Western blots are shown. PAI-1
protein levels were quantified using ImageQuant (Molecular Dynamics).
Bar graph indicates results from 3 experiments performed in triplicate,
and significant differences (P<0.05, ANOVA) are
indicated as *. Con indicates control.
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Effect of SNAP on PAI-1 Expression in RASMCs
The effect of the nitric oxide donor SNAP on PAI-1 mRNA expression
in RASMCs was also examined. Cells were pretreated with 10
µmol/L SNAP for 5 minutes followed by the addition of 100 nmol/L Ang
II for 3 hours. Treatment of cells with SNAP inhibited Ang
IIstimulated PAI-1 expression in RASMCs by 60% (Figure 5
). These results show that SNAP inhibits
PAI-1 expression to an extent similar to that observed with
natriuretic peptides. Treatment of cells
simultaneously with CNP (100 nmol/L) and SNAP (10
µmol/L) was not more effective than CNP alone in inhibiting Ang
IIstimulated PAI-1 mRNA (data not shown), suggesting that
natriuretic factors and nitric oxide may share a common
mechanistic pathway in the suppression of PAI-1.

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Figure 5. Effect of SNAP on PAI-1 expression in RASMCs.
RASMCs were pretreated with 10 µmol/L SNAP for 5 minutes
followed by the addition of Ang II (100 nmol/L) for 3 hours. PAI-1 mRNA
expression was determined by Northern blot analysis. Results
were visualized and quantified by PhosphorImage analysis.
Representative Northern blots of PAI-1 and 36B4 are
shown. Bar graph represents PAI-1 mRNA levels normalized to
36B4 from 3 experiments performed in triplicate, and significant
differences (P<0.05, ANOVA) are indicated as *. Con
indicates control.
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Role of cGMP in the Regulation of PAI-1 Expression
ANF and CNP primarily signal via natriuretic peptide
receptor/guanylyl cyclases A and B (NPR-A and NPR-B),
respectively.40 41 Nitric oxide activates
a soluble guanylyl cyclase, which is the primary mediator of its
actions in VSMCs.42 Because cGMP is a major
second messenger for both natriuretic peptides and nitric
oxide, we examined the role of cGMP in the regulation of PAI-1
expression. Intracellular cGMP was directly elevated by treatment of
cells with the membrane-permeant cGMP analogue 8-Br-cGMP for 15 minutes
followed by stimulation with 100 nmol/L Ang II for an additional 3-hour
incubation. The effect of 8-Br-cGMP on PAI-1 mRNA was determined by
Northern blot analysis as described above. Figure 6
top shows that 8-Br-cGMP decreased
PAI-1 mRNA levels in Ang IIstimulated cells in a
concentration-dependent manner. 8-Br-cGMP reduced Ang IIstimulated
PAI-1 mRNA by 60%, which is quantitatively similar to the
inhibitory effects observed with natriuretic
peptides (Figure 2
, top and bottom left) and SNAP (Figure 5
). Moreover,
the inhibition of PAI-1 levels by the combination of ANF (100 nmol/L)
with 8-Br-cGMP (1 mmol/L) was similar to that observed with ANF
alone (Figures 1
and 6
, top). These results show that the direct
elevation of intracellular cGMP levels by 8-Br-cGMP mimics the
inhibitory effects of ANF, CNP, and SNAP on PAI-1
expression.
Treatment of RASMCs with ANF, CNP, and SNAP increased cGMP levels from
5- to 10-fold (data not shown), confirming that these factors are
potent stimulators of intracellular cGMP levels in
RASMCs.40 42 The role of cGMP in SNAP's
inhibitory effect on PAI-1 expression was examined by
treating cells with a soluble guanylyl cyclase inhibitor,
ODQ, which does not inhibit adenylyl cyclase or membranous guanylyl
cyclase activity.42 ODQ prevented SNAP-induced
increases in cGMP in RASMCs (data not shown), as described
previously.42 Treatment of RASMCs with ODQ also
significantly impaired SNAP's ability to inhibit PAI-1 expression in
Ang IIstimulated RASMCs (Figure 6
, bottom left). To examine the
potential role of cGMP-dependent protein kinase (cGPK) in the
regulation of PAI-1 expression, cells were treated with KT5823, an
inhibitor of cGPK previously shown to interfere with a
variety of other actions of natriuretic factors and nitric
oxide.27 43 44 45 Pretreatment of cells with
10 µmol/L KT5823 for 10 minutes did not significantly alter Ang
II stimulation or ANF suppression of PAI-1 mRNA levels (Figure 6
, bottom right). Thus, whereas cGMP appears to play an important role in
the suppression of PAI-1 expression by natriuretic factors
and nitric oxide, this effect of cGMP does not appear to require cGPK
activity.
Effect of ANF on PAI-1 mRNA Half-life
Recent studies have demonstrated that cGMP can reduce mRNA levels
either by reducing transcription46 47 or by
decreasing transcript stability.48 The effect of
ANF on PAI-1 mRNA half-life was evaluated in RASMCs treated with
actinomycin D (ActD), an inhibitor of
transcription.48 Cells were stimulated for 1 hour
with 100 nmol/L Ang II followed by the addition of ActD in the absence
or presence of 100 nmol/L ANF. PAI-1 mRNA was measured over a time
course of 2 hours, such that the total stimulation time for the cells
with Ang II was 3 hours, as described in Figures 1
left and 2 left.
This study demonstrated that PAI-1 mRNA levels decreased in a
time-dependent manner after the addition of ActD, whereas Ang
IIstimulated cells incubated without ActD continue to increase PAI-1
mRNA over this time course (Figure 7
).
There was no significant difference in the rate of PAI-1 mRNA decline
in ActD-treated cells in the absence or presence of ANF, suggesting
that ANF does not destabilize PAI-1 mRNA.

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Figure 7. Effect of ANF on PAI-1 mRNA half-life in RASMCs.
Cells were incubated with (closed symbols) or without (open symbols)
100 nmol/L Ang II for 1 hour. Act D (10 µg/mL) and ANF (100 nmol/L)
were added as indicated and incubated for the additional times shown.
PAI-1 mRNA levels normalized to 36B4 from 3 separate experiments are
shown. Closed diamonds indicate the time course of Ang IIstimulated
PAI-1 expression in the absence of Act D or ANF.
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Discussion
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Elevated PAI-1 levels have been strongly implicated as a
risk
factor for cardiovascular disease in NIDDM,
insulin resistance,
and obesity.
8 9 11 13 Because
impaired endothelium-dependent
vasorelaxation occurs in
the insulin resistance syndrome and
NIDDM,
49 50
we investigated the potential role of cGMP-coupled
vasorelaxant
hormones, including natriuretic factors and nitric
oxide,
in the regulation of PAI-1 expression in VSMCs. This
study revealed
that ANF and CNP and the nitric oxide donor SNAP
were potent
inhibitors of PAI-1 expression in VSMCs, at both
the mRNA
and protein levels. Dose-response studies demonstrated
that similar low
nanomolar concentrations of ANF and CNP were
sufficient for the
inhibition of PAI-1 expression, suggesting
that
physiological concentrations of these
natriuretic factors
may be sufficient to reduce vascular
PAI-1 mRNA levels in vivo.
The inhibition of PAI-1 expression by these
natriuretic factors
and SNAP was mimicked by the
membrane-permeant cGMP analogue
8-Br-cGMP, and the inhibition of PAI-1
expression by SNAP was
blocked by the soluble guanylyl cyclase
inhibitor ODQ. These
results provide strong evidence that
the elevation of cGMP by
natriuretic peptides and NO
inhibits PAI-1 expression in VSMCs.
To our knowledge, this is the first
description of such a role
for vasoactive hormones, and a general
signaling pathway, in
the inhibition PAI-1 expression.
Previous reports have shown that the vasopressive peptide Ang II is a
potent stimulator of PAI-1 expression in both vascular
endothelial and SMCs23 24 51 and
that angiotensin-converting enzyme (ACE) inhibition reduces
both vascular and plasma PAI-1 levels.18 52 These
reports suggest that the renin-angiotensin system, via the
actions of Ang II, can contribute to the expression of PAI-1. However,
because ACE inhibitors also impair the breakdown of
kinins,53 increased kinin-stimulated nitric oxide
synthesis may contribute to the decreases in PAI-1 expression
associated with ACE inhibition.18 52 Thus, it
will be important to determine whether Ang II receptor
antagonists are as effective as ACE inhibitors
in reducing PAI-1 levels in vivo.
One remarkable feature of the inhibition of PAI-1 expression by
ANF, CNP, nitric oxide, and 8-Br-cGMP was that the maximal
inhibitory effectiveness of these agents was similar,
ranging from 50% to 70% inhibition. This finding was
consistently observed over a range of Ang II and PDGF
concentrations (Figure 1
, left and right) and over a time course of
natriuretic treatment (Figure 2
, bottom right). The finding
that ANF did not alter PAI-1 mRNA half-life (Figure 7
) suggests that
natriuretic factors suppress PAI-1 mRNA levels by
inhibiting transcription. This finding is consistent with other
reports that have shown that ANF can inhibit transcription of its
receptor (NPR-A) and type I cGMPdependent protein
kinase.46 Moreover, the autoregulation of NPR-A
may provide a negative feedback on ANF's inhibitory effect
on PAI-1 under conditions of chronic ANF stimulation. The observation
that natriuretic factors, SNAP, and 8-Br-cGMP did not
completely inhibit PAI-1 expression may suggest either that these
factors can block only a subset of the PDGF- and Ang IIsignaling
pathways that induce PAI-1 expression or that cGMP reduces PAI-1
transcription independently of these hormone-signaling pathways.
Studies are currently underway to identify the specific mechanism(s)
responsible for the inhibitory effects of cGMP on PAI-1
expression.
The finding that natriuretic factors and nitric oxide
inhibit PAI-1 expression in VSMCs may have important implications
related to the regulation of fibrinolysis and vascular
disease. Exogenous delivery of nitric oxide, by administration of a
nitric oxide donor, has been shown to reduce thrombus
formation.34 35 54 The suppression of PAI-1
released by platelets36 37 and by VSMCs
(current report) may contribute to this nitric oxide effect by
promoting fibrinolysis. Consistent with this
hypothesis, nitric oxide synthase inhibition has been reported to
increase plasma PAI activity in environmentally stressed
rats.55 Moreover, the suppressive effect of
nitric oxide on PAI-1 may be diminished in certain
pathophysiological states, such as obesity, insulin
resistance, and NIDDM, which display both reduced
endothelium-dependent
vasodilation49 50 and elevated PAI-1
levels.8 9
The plasminogen activator/inhibitor
system has been shown to modulate the development of vascular lesions
in injured and atherosclerotic vessels. Studies using gene knockout
mice have shown that plasminogen deficiency impairs and
PAI-1 deficiency enhances neointimal development after
arterial injury.56 57 These changes
in neointimal formation may reflect an important role of
plasmin-mediated proteolysis of the ECM in releasing constraints that
interfere with VSMC migration. In addition, PAI-1 can also directly
impair VSMC migration by binding to vitronectin and thereby
interfere with its interactions with the cellular
vitronectin receptor
Vß3 that facilitates
integrin-mediated cell migration.7 These studies
suggest that PAI-1 may reduce the stage of neointimal
development that is dependent on VSMC migration. However, although
natriuretic factors and nitric oxide suppress PAI-1
expression, these vasoactive factors also directly inhibit VSMC
migration.27 28 Therefore, it will be important
to determine whether inhibition of PAI-1 expression by these
cGMP-coupled hormones modulates their effects on cellular migration. In
contrast, plasminogen deficiency accelerates lesion
development in atherosclerosis-prone apolipoprotein
Edeficient mice.58 Thus, plasmin appears to
provide protective effects against atherosclerotic lesion formation
that may be diminished by inhibitors of
plasminogen activation, such as PAI-1. Inhibition of
vascular PAI-1 expression by natriuretic factors and nitric
oxide may promote fibrinolysis and turnover of the
ECM,1 2 6 thereby reducing advanced stages of
lesion formation.
In summary, we report that natriuretic factors, including
CNP and ANF, and nitric oxide, acting via a cGMP-dependent mechanism,
are potent inhibitors of PAI-1 expression in VSMCs. The
suppression of PAI-1 expression by these vasorelaxation factors
antagonizes the induction of PAI-1 by the vasopressive hormone Ang II,
suggesting that the combined effects of vasopressor and vasorelaxant
hormone action may influence the fibrinolytic balance.
 |
Acknowledgments
|
|---|
This work was supported in part by National Institutes of
Health
grants DK 48358 (to E.P.F) and DK 36836 (Joslin's Diabetes and
Endocrinology
Research Center Grant), and grants from the Adler
Foundation
and Juvenile Diabetes Foundation International (to
E.P.F.).
Received February 24, 1998;
accepted May 7, 1998.
 |
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