© 2000 American Heart Association, Inc.
Thrombosis |
HMG CoA Reductase Inhibitors Reduce Plasminogen Activator Inhibitor-1 Expression by Human Vascular Smooth Muscle and Endothelial Cells
From Brigham & Women’s Hospital and Harvard Medical School, Department of Medicine, Vascular Medicine and Atherosclerosis Unit, Boston, Mass.
Correspondence to Peter Libby, MD, Brigham & Women’s Hospital, Vascular Medicine & Atherosclerosis Unit, 221 Longwood Ave, LMRC-309, Boston, MA 02115. E-mail plibby{at}rics.bwh.harvard.edu
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Abstract |
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Abstract—The clinical benefit of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) may derive from a qualitative, functional change in atherosclerotic lesions in addition to their lipid-lowering properties. We examined whether statins altered expression of the major determinants of fibrinolytic balance, plasminogen activator inhibitor-1 (PAI-1), and tissue-type plasminogen activator (tPA) in human vascular smooth muscle (SMC) and endothelial (EC) cells. Simvastatin reduced levels of PAI-1 antigen released from SMCs and ECs stimulated with platelet-derived growth factor or transforming growth factor-ß (IC50
1 µmol/L).
Levels of EC-derived tPA increased 2-fold over the same concentrations
of simvastatin that inhibited release of PAI-1.
Simvastatin’s inhibitory effect was mimicked
by C3 exoenzyme and prevented by geranylgeranyl pyrophosphate, but not
by farnesyl pyrophosphate, suggesting the involvement of
geranylgeranyl-modified intermediates. Decreased PAI-1 antigen was
correlated with reduced mRNA transcription and activity of the PAI-1
promoter. By inhibiting expression of PAI-1 from SMCs and ECs while
increasing expression of tPA from ECs, simvastatin may
alter the local fibrinolytic balance within the vessel wall toward
increased fibrinolytic capacity that, in turn, would reduce thrombotic
risk after plaque rupture.
Key Words: atherosclerosis • HMG CoA reductase inhibitors • fibrinolysis • plasminogen activator inhibitor-1 • tissue plasminogen activator
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Introduction |
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Lipid-lowering treatment with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, reduces the incidence of acute cardiovascular events and lowers total mortality in patients with either high or "average" levels of cholesterol.1 2 3 Interestingly, quantitative angiographic studies of cholesterol reduction have revealed only a relatively modest (1% to 2%) change in coronary arterial stenosis in the face of substantial clinical benefit.4 Therefore, statins may improve qualitative, functional features of atheroma, which account for clinical benefits out of proportion to changes in lesion size.
Cellular mechanisms, beyond reduced cholesterol synthesis, may underlie statin’s vasculoprotective effect observed clinically. Statins reduce cellular pools of farnesyl and geranylgeranyl pyrophosphates, metabolites of mevalonate used in posttranslational prenylation of proteins.5 6 7 Reduced prenylation of GTP-binding proteins, such as Ras and Rho, may contribute to statin’s well-documented effect of inhibiting cell growth.8 9 Recent evidence shows that statins can increase expression of profibrinolytic factors while reducing expression of prothrombotic factors, effects also dependent on the inhibition of prenylated intermediates. Endothelial expression of tissue plasminogen activator (tPA) and nitric oxide is enhanced by treatment with statins, effects that would favor local cellular fibrinolysis.10 11 12 Conversely, treatment with statins inhibits macrophage expression of the prothrombotic molecule tissue factor.13 Statins may thus alter the local cellular fibrinolytic balance toward increased fibrinolytic potential and, if functional in vivo, promote antithrombotic mechanisms by cells within atheroma.
Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of the plasminogen activators tPA and urokinase in the regulation of fibrinolytic balance.14 15 Disorders associated with increased thrombosis, such as coronary heart disease, deep-vein thrombosis, and obesity, are associated with increased PAI-1 antigen and activity and, in the case of coronary heart disease, decreased tPA activity.16 17 18 Indeed, both increased plasma PAI-1 levels and a 4G/5G promoter polymorphism that leads to increased plasma PAI-1 are correlated with risk for recurrent myocardial infarction, usually a thrombotic event.19 Human atherosclerotic lesions also exhibit increases of PAI-1 protein and mRNA, localized to smooth muscle cells (SMCs) and inflammatory cells at the sides and bases of lesions, as well as to the overlying endothelium.20 21 22 23 These results suggest that local synthesis of PAI-1 and its balance with plasminogen activators may regulate regional plasmin generation and, in turn, influence the regional fibrinolytic balance within atheroma. In this study, we investigated whether statins modulate expression of PAI-1 and tPA elaborated by vascular SMCs and endothelial cells (ECs) exposed to inflammatory stimuli. We further investigated potential molecular mechanisms that underlie decreased PAI-1 expression.
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Methods |
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Materials
Simvastatin was provided by Merck & Co (West Point, Pa) and activated by alkaline hydrolysis of the lactone moiety, according to the manufacturer’s protocol. Human recombinant platelet-derived growth factor (PDGF)-BB was from Genzyme; human platelet-derived transforming growth factor (TGF)-ß1 was from Collaborative Biomedical Products. Farnesyl pyrophosphate (Fpp), geranylgeranyl pyrophosphate (GGpp), and C3 exoenzyme were purchased from Biomol.
Cell Culture
SMCs obtained from explanted sections of human saphenous vein
were grown in Dulbecco’s modified Eagle’s medium (DMEM; Life
Technologies, Inc) supplemented with 20 mmol/L HEPES, 10% fetal
calf serum (Hyclone), and 5 mmol/L L-glutamine in a
humidified atmosphere of 5% CO2/95% air. ECs
were derived from human saphenous veins as described
previously.24 For experiments, SMCs were placed in
serum-free DMEM/Ham’s F12 for 24 hours to reduce exposure to serum
components. Cells were then pretreated for an additional 16 hours with
various statins in 1.0 mL serum-free medium before challenge with
various cytokines. ECs were cultured in serum-free medium 199
containing 0.2% human serum albumin with or without statins
for 16 hours and then stimulated with cytokines in 1.0 mL of
fresh, serum-free medium.
Western Immunoblot and ELISA Analysis
After the experiments, conditioned media were collected and
divided in 2 aliquots: 1 was mixed 4:1 with 5x loading buffer (1x is
125 mmol/L Tris-HCl, pH 6.8; 10% glycerol; and 2% SDS)
and boiled for immunoblot analysis; the other
aliquot was used directly for ELISA. The remaining cell layer was
dissolved in 0.1% SDS/0.1N NaOH and assayed for protein content by the
bicinchoninic acid method (Pierce Chemical Co). Western
immunoblotting was performed with mouse monoclonal
antibodies to human PAI-1 or tPA (diluted 1:250, American
Diagnostica) and horseradish peroxidase–conjugated rabbit
anti-mouse IgG as a secondary antibody diluted 1:15 000. ELISA for
human PAI-1 (recognizing latent, active, and complexed forms) and for
total tPA was performed by using a commercially available kit (American
Diagnostica).
Metabolic Labeling and Immunoprecipitation
To monitor de novo synthesis of PAI-1, cells cultured in 6-well
dishes were incubated for 24 hours in 1.0 mL DMEM lacking cysteine and
methionine but supplemented with 50 µCi/mL 35S
protein labeling mix (NEN). After cell stimulation, 200 µL of
conditioned medium was collected and precleared with normal mouse serum
and protein A–agarose conjugate, followed by addition of 2 µg of
mouse monoclonal 
–PAI-1 antibody (Oncogene Science, AB3; 2 hours at
4°C) and precipitation with protein A–agarose. Nonimmune mouse IgG
was used as a control. Immune precipitates were washed 4 times in
buffer consisting of 10 mmol/L Tris-HCl (pH 7.4), 150 mmol/L
NaCl, 1% Triton X-100, 0.5% NP-40, and 1.0 mmol/L EDTA, and the
pellets were resuspended in 2x SDS lysis buffer (1x is 125
mmol/L Tris-HCl, pH 6.8; 10% glycerol; 2% SDS; and 5%
2-mercaptoethanol). Samples were boiled for 5 minutes and
centrifuged, and supernatants were fractionated by 10%
SDS–polyacrylamide gel electrophoresis. The gels were dried
and subjected to autoradiography. Total protein
synthesis was assessed by the levels of
[35S]methionine/cysteine incorporated into
trichloroacetic acid–precipitable material from lysates of the
remaining cell layers.
RNA Isolation and Northern Blot Analysis
Total RNA was isolated with RNazol (Tel-Test) from
statin-treated human SMCs cultured in 100-mm2
dishes after stimulation with agonists for the indicated times. Ten
micrograms of total RNA was fractionated on 1.2% agarose/2.2 mol/L
formaldehyde gels and blotted to Hybond nylon filters by capillary
transfer. Forty–base pair oligonucleotide probes to
human PAI-1 or GAPDH (Oncogene Sciences) were end-labeled with
[
32P]ATP and T4 polynucleotide
kinase and purified through G-25 Sephadex columns. Blots were
hybridized overnight at 42°C with 2 to 10x106
counts per minute of probe in buffer consisting of 0.5 mol/L
NaPO4 , pH 7.2; 7% SDS; and 1% BSA. Blots were
washed and exposed to autoradiography film for 1 to 3
days in a cassette containing 2 intensifying screens.
Nuclear Isolation and Run-On Analysis
Human SMCs from 4 confluent 175-cm2 flasks
per experimental group were collected by brief trypsinization and
centrifuged. Cell pellets were resuspended in 5.0 mL ice-cold
lysis buffer (10 mmol/L Tris-HCl, pH 7.4; 10 mmol/L NaCl;
3 mmol/L MgCl2; and 0.5% NP-40), gently
triturated, and collected by centrifugation
(1000g, 5 minutes). Cell pellets were subjected to 3
additional rounds of resuspension and trituration in lysis buffer. An
aliquot stained with trypan blue verified lysis of the cells and
revealed intact nuclei largely free of cytoplasmic membranes. Nuclear
pellets were resuspended in 100 µL of storage buffer (20 mmol/L
Tris-HCl, pH 8.1; 75 mmol/L NaCl; 0.5 mmol/L EDTA; 1
mmol/L DTT; and 50% glycerol) and stored at -80°C.
In vitro transcription was performed by mixing 100 µL of nuclei with
100 µL of transcription buffer containing 10 mmol/L Tris-HCl, pH
8.0; 5 mmol/L MgCl2; 300 mmol/L KCl;
50 µmol/L EDTA; 1 mmol/L DTT; 250 U/mL RNasin; and 0.5
mmol/L each of CTP, ATP, GTP, and 200-µCi
[32P]UTP. Reactions were incubated at 30°C
for 30 minutes and terminated by addition of 20 U of RNase-free DNase
I. After proteinase K digestion and phenol/chloroform extraction, newly
transcribed mRNA was precipitated with ammonium acetate and 2.5 volumes
of ice-cold ethanol. RNA pellets were briefly dried and resuspended in
500 µL of hybridization buffer. Radiolabeled RNA
(107 cpm) was hybridized for 48 hours at
42°C to cDNAs (1 µg) from human PAI-1, ß-tubulin, and pGEM vector
fixed on Hybond nylon filters. After being washed in 1x SSC, filters
were exposed to autoradiography film for 1 to 3 days in
cassettes with 2 intensifying screens.
Transient Transfection
The human PAI-1 promoter constructs p800, p187, and p100 that
contain the luciferase reporter have been described and were generously
provided by Dr David Loskutoff.25 Bovine aortic SMCs
cultured in 12-well dishes were transfected with 0.25 µg of each
reporter construct by the lipofectamine-plus method for 5 hours in
Optimized minimal essential medium (Gibco-BRL). Efficiency of
transfection (12% to 18%) was not altered by experimental treatments,
as assessed by expression of green fluorescent protein directed
by the cytomegalovirus promoter. This approach was necessary because of
increased ß-galactosidase activity (40%) in bovine aortic
SMCs exposed to PDGF without a change in cell number, thus invalidating
the use of this reporter for assessing changes in transfection
efficiency. After transfections, cells were incubated with
simvastatin for 16 hours in serum-free DMEM/F12 medium and
then stimulated with PDGF-BB (30 ng/mL) for an additional 18 hours.
Cell lysates were collected and luciferase activity measured in an
aliquot by using a Lumat LB 9501 luminometer. In any individual
experiment, values of luciferase activity from triplicate wells varied
<10%.
Statistical Analysis
Statistical differences among groups were tested by Students’
t test (2 groups) or by 1-way ANOVA (>2 groups). Values are
presented as the mean±SEM. A probability value 
0.05 was
considered significant.
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Results |
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Statins Reduce PAI-1 Elaborated by Vascular SMCs and ECs and Increase tPA Released by ECs
Initial experiments investigated whether statins could alter the levels of PAI-1 released by human vascular SMCs or ECs. Two inducers of PAI-1 expression, PDGF and TGF-ß, increased levels of PAI-1 released from SMCs by 2.6±0.3- and 2.7±0.3-fold, respectively. Simvastatin, in a concentration-dependent manner, significantly reduced the effect of PDGF and TGF-ß on PAI-1 levels (Figure 1A
). Immunoblot
analysis of conditioned medium confirmed the presence of a
47-kDa band that was increased by stimulation with PDGF and TGFß and
was reduced by increasing concentrations of simvastatin
(Figure 1A).
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In ECs, TGF-ß increased levels of PAI-1 by 2.0±0.1-fold. This
increase was also reduced by simvastatin in a
concentration-dependent manner (Figure 1B
). The
plasminogen activator tPA was detected in
medium conditioned by ECs but not by SMCs and was not altered by
TGF-ß stimulation. Of interest, simvastatin increased
EC-derived tPA to a maximum of 2-fold over controls at concentrations
that inhibited induction of PAI-1 (Figure 1B). Under the
serum-free conditions of the experiments, tPA antigen in
SMC-conditioned medium was below the detection limit of the ELISA
employed (1 to 2 ng/mL). Immunoblot analysis of
PAI-1 and tPA confirmed the results obtained by ELISA.
To verify that simvastatin reduced synthesis of PAI-1
protein, SMCs and ECs were metabolically labeled, and PAI-1
was immunoprecipitated from the medium. Simvastatin reduced
de novo synthesis of PAI-1 protein by SMCs stimulated by PDGF and
TGF-ß and by ECs stimulated by TGF-ß, without inhibiting total
protein synthesis (Figure 2). An
additional protein of 70 kDa from SMCs (visible after longer
exposure times) and a protein of 50 kDa from ECs coprecipitated with
PAI-1 and remained constant across experimental groups, verifying that
changes in PAI-1 were not due to different levels of loading. It is not
known whether these coprecipitating proteins specifically interact with
PAI-1 or represent low-affinity interactions with the
PAI-1/antibody immune complex. Thus, statins reduced the levels of
PAI-1 synthesized from cytokine-stimulated human SMCs and ECs
while increasing the levels of tPA released from ECs.
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Geranylgeranyl but Not Farnesyl Isoprenoids Reverse
Simvastatin’s Inhibitory Effect
Experiments with the substrate of 3-hydroxy-3-methylglutaryl
coenzyme A reductase, mevalonate, and the isoprenoid intermediates GGpp
and Fpp evaluated their involvement in simvastatin’s
inhibitory effects. Mevalonate and GGpp, but not Fpp,
prevented simvastatin-induced inhibition of PAI-1 (Figure 3A). Under the same conditions, the
uptake of tritiated GGpp and Fpp by the cultured SMCs was similar, thus
excluding a difference in cellular entry of the isoprenes as an
explanation for Fpp’s lack of effect (data not shown). The Rho family
of small GTP-binding proteins are posttranslationally modified by
geranylgeranylation and have been implicated in statin’s mechanism of
inhibiting the expression of tissue factor and increasing the
expression of type I nitric oxide synthase and
tPA.10 12 13 Human SMCs were therefore treated with C3
exoenzyme (15 µg/mL), a transferase that selectively ADP-ribosylates
and thus inactivates Rho proteins. We compared the effect
of this treatment to that of simvastatin on PAI-1
expression. C3 exoenzyme inhibited PDGF-induced increases of PAI-1,
thus reproducing the inhibitory effect of
simvastatin (Figure 3B). The addition of GGpp did
not reverse the inhibitory effect of C3 exoenzyme,
consistent with its mechanism of Rho inactivation.
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Statins Reduce PAI-1 Gene Transcription
To explore further the mechanism of statin’s
inhibitory effect, Northern blot analysis and
nuclear run-on assays were performed to determine whether statins
reduced PAI-1 transcription. PDGF stimulation of human SMCs increased 2
PAI-1 transcripts (3.2 and 2.5 kb) in a time-dependent manner
(Figure 4A). Preincubation with
simvastatin prevented accumulation of both PAI-1
transcripts. PDGF-induced increases of PAI-1 mRNA were in part due to
enhanced gene transcription, which increased 3.2±0.8-fold, on average
(n=3), after correction for levels of ß-tubulin (Figure 4B).
Simvastatin alone had a modest effect and significantly
(P0.05) inhibited PDGF-induced increases of PAI-1
transcription. Thus, statins reduce PAI-1 protein expression in part by
reducing transcription of the PAI-1 gene.
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Statins Reduce Activity of the PAI-1 Promoter
Reduced PAI-1 gene transcription suggests that statins may inhibit
activity of the PAI-1 promoter. Transient transfection experiments were
performed with an 800-bp region of the PAI-1 promoter, previously shown
to be sufficient for inducibility by phorbol ester and
TGF-ß.26 27 Bovine aortic SMCs were used for these
experiments, as they have a greater efficiency and reproducibility of
transfection compared with human SMCs. Luciferase activity from
construct p800, which encompasses -800 to +71 bp of the human PAI-1
promoter, increased 3.3-fold in response to PDGF. Deleting promoter
segments to positions -187 (p187) or -100 (p100) did not reduce the
fold induction in response to PDGF (3.3- and 4.2-fold, respectively),
but absolute luciferase activity decreased with shorter promoter
fragments (Figure 5).
Simvastatin significantly reduced PDGF-induced increases of
luciferase activity from p800, p187, and p100. Of note, the
inhibitory effect of simvastatin on
PDGF-induced activity of p100, though significant, was less pronounced
than on the p800 and p187 constructs. Transfection with promoterless
vector (p19-Luc) showed background levels of luciferase activity across
the experimental groups. Statin-induced inhibition of PAI-1 gene
transcription may thus stem from reduced activity of the PAI-1
promoter.
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Discussion |
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Clinical trials of statin therapy have demonstrated a reduced incidence of cardiovascular events in the face of relatively modest angiographic changes, suggesting that effects of statins other than on the degree of arterial stenosis may underlie part of their clinical benefit. Increased levels of PAI-1 both systemically and locally within human atheroma may contribute to the hypercoagulable state noted in hypercholesterolemic individuals and may predispose such subjects to an increased risk of thrombotic complications. This study investigated whether statins alter vascular cell expression of PAI-1 and tPA, the major regulatory proteins of fibrinolytic balance, that may in turn promote increased fibrinolysis.
Statins inhibited expression of PAI-1 from human SMCs and ECs exposed
to PDGF or TGF-ß and increased expression of tPA from ECs. The
results with human ECs support those of Essig et al,10 who
recently reported increased tPA and reduced PAI-1 expression by a
transformed rat EC line after treatment with statins. We show here that
statins inhibit expression of PAI-1 by human SMCs through a mechanism
involving reduced activity of the PAI-1 promoter and transcription of
the PAI-1 gene. The concentrations of statins that inhibited expression
of PAI-1 (0.5 to 2 µmol/L) correspond closely with those
reported to inhibit other cellular functions, such as proliferation and
expression of tissue factor,8 13 but are well below those
reported to induce apoptosis of vascular SMCs (
10
µmol/L).28 Concentrations of simvastatin
within vascular tissue in vivo have not been measured; however, peak
plasma concentrations after administration of 40 mg
simvastatin (0.1 to 0.2 µmol/L) approach those
required to inhibit cellular functions in vitro.29 The
current data suggest that statins indeed affect expression of proteins
central to the regulation of fibrinolytic balance, independent of lipid
lowering in part via reduced transcription.
The reversal of simvastatin’s inhibitory effect by mevalonate and GGpp, but not by Fpp, indicates the requirement of geranylgeranylated intermediates in the pathway of PAI-1 expression. The Rho family of GTP-binding proteins are posttranslationally modified in this manner and regulate several cell functions, including cytoskeletal organization, motility, and growth.30 C3 exoenzyme, which inactivates Rho and Rac by ADP ribosylation,31 mimicked simvastatin’s inhibitory effect, implicating Rho family members in the mechanism of PDGF-induced expression of PAI-1. Of note, a dominant/negative Rac construct inhibited TGF-ß induction of the PAI-1 promoter in 3T3 fibroblasts, whereas dominant/negative constructs of Ras, Cdc42, and Rho did not, suggesting a key role of Rac in PAI-1 expression.32 Moreover, inactivation of Rho proteins may also underlie statin-mediated enhancement of type I nitric oxide synthase and tPA expression by ECs and inhibition of tissue factor by macrophages.10 12 13 Because several Rho proteins are inactivated by C3 exoenzyme, additional studies are needed to elucidate which family members contribute to induction of PAI-1 expression.
Simvastatin reduced PDGF-induced transcriptional activity of the PAI-1 gene, as assessed by nuclear run-on analysis. This inhibition likely results from corresponding reductions in the activity of the PAI-1 promoter after treatment with simvastatin. Although absolute promoter activity declined with shorter promoter fragments, PDGF inducibility remained constant down to 100 bp upstream of the transcriptional start site. Simvastatin inhibited PDGF induction of all promoter constructs tested; however, the inhibitory effect on p100, the smallest promoter segment tested, was less pronounced than on the 2 larger promoter constructs, p187 and p800. How simvastatin inhibits PAI-1 promoter activity is not resolved, but the data suggest that simvastatin reduces the activity or binding of transcription factors to sequences in the proximal 187 bp of the PAI-1 promoter. This region encompasses a cAMP response element/activator protein-1 element (or p-box) that resembles the consensus site for both activator protein-1 and cAMP response element binding proteins (TGAGTTCA) and is necessary for promoter activity induced by phorbol ester and serum.26 33 Further deletions of the PAI-1 promoter showed that the p-box element is also required for PDGF inducibility (data not shown). However, simvastatin reduced PAI-1 expression induced by several agonists, including interleukin-1, angiotensin II, and TGF-ß, the latter utilizing promoter elements distinct from the p-box.34 This suggests a common mechanism whereby simvastatin decreases PAI-1 promoter activity independent of the inducing stimulus and associated transcription factors.
Despite the incompletely resolved mechanism by which statins reduce expression of PAI-1 from SMCs and ECs while increasing tPA from ECs, the consequences of this finding have particular medical interest. Reducing local expression of PAI-1 by SMCs within vascular lesions while increasing tPA expression by luminal ECs may tip the local fibrinolytic balance toward increased fibrinolytic capacity, which could limit the extent of thrombus formation and fibrin(ogen) deposition that follows plaque rupture. On the other hand, increased local fibrinolysis may also promote extracellular matrix degradation that in turn may destabilize advanced atherosclerotic plaques. Treatment of humans with statins has inconsistent effects on circulating PAI-1 antigen35 ; however, such clinical studies cannot assess local fibrinolytic balance within the vessel wall. Altering the local fibrinolytic balance of SMCs or ECs toward increased fibrinolysis may reduce thrombotic risk, thus contributing to the clinical benefit conferred by treatment with statins.
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Acknowledgments |
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This work was supported by National Institutes of Health grant R37 HL34636-13 (to P.L.). Dr T. Bourcier is a recipient of National Research Service award HL09483-01. We thank Dr David Loskutoff (Research Institute of Scripps Clinic, La Jolla, Calif) for the PAI-1 promoter constructs and Elissa Simon-Morrisey and Dr Marysia Muszynski (Brigham & Womens Hospital, Boston, Mass) for their excellent and skillful assistance.
Received April 12, 1999; accepted August 11, 1999.
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