Full text of DOI:10.1038/sj.bjp.0704454

British Journal of Pharmacology (2002) 135, 284 ± 292
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HMG CoA reductase inhibitors a€ect the ®brinolytic system of
human vascular cells in vitro: a comparative study using
di€erent statins
1
1
1
1
1Franz Wiesbauer, Christoph Kaun, Gerlinde Zorn, Gerald Maurer, Kurt Huber &
*,1Johann Wojta
1Department of Internal Medicine II, University of Vienna, Austria
1
The results of several clinical studies investigating the e€ect of statin therapy on the ®brinolytic
system in vivo are inconclusive. We compared the e€ect of six di€erent statins (atorvastatin,
cerivastatin, ¯uvastatin, lovastatin, pravastatin, simvastatin) on components of the ®brinolytic
system expressed by human vascular endothelial cells and smooth muscle cells and by the human
hepatoma cell line HepG2.
2
All statins used except pravastatin signi®cantly decreased PAI-1 production in human endothelial
and smooth muscle cells. This e€ect was also seen in the presence of IL-1a and TNF-a. All statins
except pravastatin increased t-PA production in human smooth muscle cells. On a molar basis
cerivastatin was the most e€ective HMG CoA reductase inhibitor used. Only simvastatin and
lovastatin increased t-PA production in endothelial cells. The e€ects on the ®brinolytic system were
reversed by mevalonate. Statins decreased mRNA levels for PAI-1 in endothelial and smooth muscle
cells and increased mRNA levels for t-PA in smooth muscle cells. Statins did not a€ect PAI-1
expression in HepG2 cells. Cell viability was not in¯uenced by statins in endothelial cells and HepG2
cells whereas in smooth muscle cells a cytotoxic e€ect was seen at high concentrations.
3
If the e€ects on the ®brinolytic system of vascular cells in vitro shown in this study are also
operative in vivo one could speculate that by increasing t-PA and decreasing PAI-1 at sites of
vascular lesions statins might reduce ®brin formation and thrombus development. Such an e€ect
might contribute to the clinically proven bene®ts of statin therapy.
British Journal of Pharmacology (2002) 135, 284 ± 292
Keywords: Experimental; vasculature; cellular; pathophysiology; atherosclerosis; cytokines; endothelial function; smooth
muscle; thrombosis
Abbreviations: BSA, bovine serum albumin; GAPDH, glyceraldehyde-3-phopshate dehydrogenase; HCASMC, human coronary
artery smooth muscle cells; HMG CoA, 3-hydroxy-3methylglutaryl-coenzyme A; HSMEC, human skin
microvascular endothelial cells; HUVEC, human umbilical vein endothelial cells; HUVSMC, human umbilical
vein smooth muscle cells; LDH, lactate dehydrogenase; MCP-1, monocyte chemotactic protein-1; MMP, matrix
metalloprotease; PAI-1, plasminogen activator inhibitor-1; SCS, supplemented calf serum; TF, tissue factor; t-
PA, tissue type-plasminogen activator; u-PA, urokinase type-plasminogen activator
Introduction
The discovery of 3-hydroxy-3-methylglutaryl-coenzyme A
reductase inhibitors or statins has led to signi®cant
improvements in the management of hypercholesteremia.
Several studies have shown that statin therapy reduces not
only the incidence of cardiovascular events but also decreases
mortality in patients with high or `average' cholesterol levels
(Sacks et al., 1996; Scandinavian Simvastatin Survival Study
Group, 1994; Shepherd et al., 1995). However recently a
signi®cant number of investigations have given evidence that
beyond their lipid lowering capacity statins exert `pleiotropic'
actions resulting in vasoprotection. Such vasoprotective
e€ects include the inhibition of smooth muscle cell growth
and the inhibition of neointima formation, the induction of
apoptosis in smooth muscle cells, the reduction of leukocyte
adhesion to and transmigration through endothelial cells, the
induction of endothelial nitric oxide synthase and the
inhibition of endothelin and monocyte chemotactic protein-
1 (MCP-1) expression in endothelial cells and the inhibition
of MCP-1, tissue factor (TF) and matrix metalloprotease-9
(MMP-9) expression in macrophages (Sindermann et al.,
2000; Stark et al., 1998; Indol® et al., 2000; Kimura et al.,
1997; Dunzendorfer et al., 1997; Laufs et al., 1997;
Hernandez-Perera et al., 1998; Romano et al., 2000; Bellosta
et al., 1998a; Ferro et al., 2000; for recent reviews see also
Rosenson & Tangney, 1998; Vaughan et al., 2000).
Recently, two studies have shown e€ects of lovastatin and
simvastatin, respectively, on the ®brinolytic system of
cultured smooth muscle cells and endothelial cells. In these
studies a decrease in plasminogen activator inhibitor type-1
(PAI-1) in both cell types was observed whereas tissue type
PA (t-PA) was increased in endothelial cells (Essig et al.,
*Author for correspondence at: Department of Internal Medicine II,
University of Vienna, A-1090 Vienna, Waehringer Guertel 18-20,
Austria; E-mail: johann.wojta@univie.ac.at
Parts of this work have been presented in oral form at the 49th
Annual Scienti®c Session of the American College of Cardiology at
Anaheim, California, U.S.A., March 12 ± 15, 2000

F. Wiesbauer et al
Statins affect the fibrinolytic system of vascular cells
285
1998; Bourcier
&
Libby, 2000). PAI-1 is the major
pieces of human umbilical cords obtained after normal
vaginal delivery (Wojta et al., 1993). Human coronary artery
smooth muscle cells (HCASMC) were isolated by the same
technique from pieces of human coronary arteries obtained
from explanted recipients' hearts after heart transplantation.
Such smooth muscle cells were cultured and characterized as
described (Wojta et al., 1993). All cells used in this study
were between passage 2 and 4. The human hepatoma cell line
HepG2 obtained from American Type Culture Collection
(ATCC; Rockville, MD, U.S.A.) was cultivated as described
(Wojta et al., 1994). All human material was obtained and
processed according to the recommendations of the hospital's
Ethics Committee and Security Board.
physiological inhibitor of t-PA and urokinase type PA (u-
PA) and therefore plays a key role in the regulation of
®brinolysis (Loskuto€ et al., 1993). High PAI-1 plasma levels
and decreased levels of t-PA activity have been shown to be
associated with coronary heart disease and PAI-1 mRNA has
been found in human atherosclerotic lesions underlining its
role in the development of these disorders (Aznar & Estelles,
1994; Schneiderman et al., 1992). Several in vivo studies have
also investigated the e€ect of statin therapy on this key
regulator of the ®brinolytic system. The results, however, are
inconclusive. Pravastatin decreased PAI-1 plasma levels in
several studies whereas atorvastatin was reported to increase
PAI-1 in plasma (Avellone et al., 1994; Wada et al., 1992;
Dangas et al., 2000; Davidson et al., 1997). For both
¯uvastatin and lovastatin an increase and a decrease in
PAI-1 levels in plasma was reported in di€erent clinical
investigations (Davidson et al., 1997; Bevilacqua et al., 1997;
Leren et al., 1988; Kostner et al., 1989; Tan et al., 1999;
Isaacsohn et al., 1994). No data are currently available on the
e€ect of cerivastatin on PAI-1 in vivo. As pointed out in a
recent review experimental/clinical disparities such as these
raise questions about the clinical applicability of in vitro
studies performed with isolated statins (Vaughan et al., 2000).
In this study we have for the ®rst time compared the e€ect of
six di€erent statins on the ®brinolytic system of human
vascular smooth muscle cells, endothelial cells and hepato-
cytes in vitro.
Treatment of cells with statins
Endothelial cells were incubated in Medium 199 (M199;
Sigma) containing 1.25% supplemented calf serum (SCS;
Hyclone, Logan, UT, U.S.A.) with di€erent concentrations of
the respective statin for the indicated time periods. Smooth
muscle cells and HepG2 cells were incubated in M199
containing 0.1% bovine serum albumin (BSA; Sigma) for
24 h prior to treatment with statins. Thereafter the medium
was replaced with fresh M199 containing 0.1% BSA, statins
were added at the concentrations indicated and the cells were
incubated for various time periods. After incubation, the
culture supernatants were collected following removal of cell
debris by centrifugation and stored at 7708C until used. The
total cell number of the respective cultures after trypsinisa-
tion was counted with a haemocytometer.
Methods
Tissue type-plasminogen activator (t-PA) antigen and
plasminogen activator inhibitor-1 (PAI-1) antigen assays
Materials
Stock solutions of atorvastatin (kindly provided by P®zer,
Sandwich, U.K.), cerivastatin (kindly provided by Bayer,
Wuppertal, Germany) and ¯uvastatin (kindly provided by
Novartis, Basel, Switzerland) were prepared at a concentra-
tion of 10 mM in ethanol. A stock solution of pravastatin
(kindly provided by Bristol-Myers-Squibb, Paris, France) at
the same concentration was prepared in distilled water. Stock
solutions (10 mM) of lovastatin and simvastatin (kindly
provided by Merck Sharpe and Dome, Ballydine, Ireland)
were prepared after activation of the molecules as described
(Cutts & Melnykovych, 1988). Such stocks were aliquoted
and stored at 7708C. Mevalonate was purchased from Sigma
(St. Louis, MO, U.S.A.). Human recombinant interleukin 1-a
(IL-1a) and human recombinant tumor necrosis factor-a
(TNF-a) were purchased from R&D Systems (Minneapolis,
MN, U.S.A.).
t-PA and PAI-1 antigen in conditioned media were determined
by speci®c enzyme-linked immunosorbent assays (ELISAs)
using monoclonal antibodies (Technoclone, Vienna, Austria).
The PAI-1 ELISA measures active, complexed and latent PAI-
1. The t-PA ELISA measures free and complexed t-PA.
Northern blots
Total cellular RNA was prepared by the guanidinium
thiocyanate-phenol-chloroform extraction from con¯uent
cells treated as indicated. RNA samples were electrophoresed
in 1.2% agarose gels, transferred to
a
Duralon-UVTM
membrane (Stratagene, U.S.A.). Hybridizations were per-
formed overnight in 50 mM PIPES, 100 mM NaCl, 50 mM
sodium phosphate, 1 mM EDTA, 5% SDS, containing
106 c.p.m./ml of the 32P labelled cDNA probes for human
PAI-1, t-PA or rat glyceraldehyde-3-phosphate dehydrogen-
ase (GAPDH). After hybridization the membranes were
washed in 5% SDS, 16SSC at room temperature for 10 min,
then washed in the same bu€er at 578C for 20 min three
times. Autoradiography was performed with XAR-5 X-ray
®lms (Eastman Kodak, U.S.A.) at 7708C (Zhang et al.,
1997).
Cell culture
Human umbilical vein endothelial cells (HUVEC) were
isolated from fresh umbilical cords by mild collagenase
treatment and cultured and characterized as described (Wojta
et al., 1992). Human skin microvascular endothelial cells
(HSMEC) were isolated from skin biopsies by trypsin
digestion and the use of Dynabeads1 (Dynal, Norway)
coated with Ulex europaeus agglutinin as described (Wojta et
al., 1992). Human umbilical vein smooth muscle cells
(HUVSMC) were isolated by the explant technique from
Determination of cell viability
In order to determine possible cytotoxic e€ects of the statins
used, lactate dehydrogenase (LDH) leakage was measured in
British Journal of Pharmacology vol 135 (1)

286
F. Wiesbauer et al
Statins affect the fibrinolytic system of vascular cells
cultures treated with statins as described above using a
commercially available assay for photometric determination
of LDH activity (Sigma).
except pravastatin decreased PAI-1 production and increased
t-PA production in HUVSMC concentration dependently
(Figure 1). Whereas atorvastatin, ¯uvastatin, lovastatin and
simvastatin showed similar concentration response curves, the
concentration response curve for cerivastatin was shifted to
the left indicating that cerivastatin could be used at
signi®cantly lower concentrations to obtain e€ects comparable
to the other statins. A time course for the e€ect of ¯uvastatin
is shown in the respective insets. A similar reduction in PAI-1
production after statin treatment was seen in HCASMC
(Table 2). At concentrations between 0.1 and 5.0 mM all statins
with the exception of pravastatin decreased PAI-1 production
concentration dependently in HUVEC after 40 h (Figure 2).
Again cerivastatin was the most e€ective when compared to
the other statins on a molar basis. A time course for the e€ect
of ¯uvastatin is shown in the inset of Figure 2. Similar e€ects
were seen when HSMEC were treated with statins (Table 2).
In contrast to the results obtained with smooth muscle cells,
upon incubation with statins only a slight increase in t-PA
production in endothelial cells was observed which reached
signi®cance only in the case of simvastatin and lovastatin
(Table 2). No e€ect of statins on u-PA production by either
smooth muscle cells or endothelial cells was seen (data not
shown). As can be seen from Figure 3 the e€ect of the
respective statins was also evident on the level of speci®c
mRNA expression. All statins tested reduced PAI-1 mRNA in
HUVSMC and HUVEC and increased t-PA mRNA levels in
HUVSMC.
Statistical analysis
Data were compared statistically by performing a Student's t-
test for paired observations unless otherwise stated in the
legend to the respective ®gure. Values of P40.05 were
considered signi®cant.
Results
Effect of statins on cell viability
As can be seen from Table 1, cell viability as determined by
leakage of lactate dehydrogenase was not signi®cantly altered
by statins in HUVEC and HepG2 whereas a cytotoxic e€ect
was observed at a concentration of 5 mM of the respective
statins in HUVSMC. At lower concentrations cell viability of
HUVSMC was not a€ected by the statins used.
Statins affect the expression of PAI-1 and t-PA in smooth
muscle cells and endothelial cells
When HUVSMC were treated for 40 h with statins at
concentrations between 0.1 and 2.5 mM, all statins tested
Table 1 E€ect of statins on viablity of human umbilical vein smooth muscle cells (HUVSMC), human umbilical vein endothelial cells
(HUVEC)and HepG2 cells
HUVSMC
HUVEC
(5.0 mM)
HepG2
(5.0 mM)
(2.5 mM)
(5.0 mM)
(100.0 mM)
Atorvastatin
Cerivastatin
Fluvastatin
Lovastatin
Pravastatin
Simvastatin
231.3+10.5**
257.9+17.3**
218.5+20.2**
290.7+24.6**
n.d.
102.2+5.7
107.6+8.1
105.9+3.7
97.7+8.7
n.d.
102.7+12.5
103.9+9.7
108.5+8.2
103.5+4.5
97.9+7.8
94.1+3.5
97.1+5.6
97.9+7.3
99.4+4.3
97.5+4.6
99.3+4.3
n.d.
n.d.
95.2+6.8
101.2+7.9
100.5+3.4
98.7+5.1
282.9+7.4**
101.5+11.2
107.8+9.6
Con¯uent monolayers of the respective cell type were incubated for 48 h in the presence or absence of statins at the indicated
concentration. Thereafter lactate dehydrogenase leakage as an indicator for cell damage was measured photometrically using a
commercially available assay as described in the Methods section. Absorbance values obtained with untreated control cells were set as
100%. Values of statin treated cells are expressed as per cent of control and represent mean values+s.d. of three independent
determinations. **P50.01; n.d.: not determined.
Table 2 E€ect of statins on PAI-1 production in human coronary artery smooth muscle cells (HCASMC), in human skin
microvascular endothelial cells (HSMEC) and in HepG2 cells and on t-PA production in human umbilical vein endothelial cells
(HUVEC)
PAI-1 antigen
HSMEC
t-PA antigen
HUVEC
HCASMC
HepG2 #1
HepG2 #2
Control
856.2+90.4
n.d.
254.9+22.1**
387.1+49.3*
n.d.
825.4+75.8
426.1+56.8*
275.5+20.8
98.5+9.3
102.9+11.3
97.1+8.8
112.9+11.2
109.9+12.8
96.3+17.6
105.3+17.8
103.3+12.3
n.d.
n.d.
103.8+8.7
101.2+10.2
104.9+7.8
102.2+13.5
1.03+0.12
1.15+0.20
1.17+0.07
1.13+0.05
1.57+0.05**
1.09+0.07
1.65+0.13**
Atorvastatin
Cerivastatin
Fluvastatin
Lovastatin
Pravastatin
Simvastatin
148.4+12.3*
125.6+14.7**
117.8+11.3**
150.3+17.5*
277.7+20.9
134.3+16.1**
Con¯uent monolayers of the respective cell type were incubated for 40 h in the absence or presence of the respective statin at a
concentration of 1.0 mM (HCASMC), 5.0 mM (HSEMC, HepG2 #1 and HUVEC) or 100 mM HepG2 #2). Conditioned media of such
treated cells were collected and PAI-1 and t-PA antigen was determined as described in the Methods section. Values are given in ng/104
cells/40 h and represent mean values+s.d. of three independent determinations. n.d.: Not determined, **P50.01, *P50.05.
British Journal of Pharmacology vol 135 (1)

F. Wiesbauer et al
Statins affect the fibrinolytic system of vascular cells
287
Statins affect the production of PAI-1 in smooth muscle
cells and endothelial cells activated by cytokines
When HUVSMC and HUVEC were treated with cytokines
such as interleukin-1a (IL-1a) or tumor necrosis factor-a
(TNF-a) an increase in PAI-1 production was seen. When
such cells were treated with these cytokines in the presence of
statins PAI-1 levels were reduced almost to levels seen in cells
incubated with statins alone (Figure 4).
Mevalonate reverses the effects of statins on PAI-1 and
t-PA in smooth muscle cells and endothelial cells
As can be seen from Table 3 the e€ects of the statins were
reversed by addition of 100 mM mevalonate. When mevalo-
nate was present during the incubation period of 40 h
together with the respective statin, PAI-1 as well as t-PA
levels were not di€erent from levels seen in untreated cells.
Statins do not affect PAI-1 in HepG2 cells
Neither of the statins used at 5.0 or 100.0 mM did a€ect PAI-
1 production in the human hepatoma cell line HepG2 after
40 h of incubation (Table 2).
Discussion
HMG CoA reductase inhibitors or statins have been shown to
exert various `vasoprotective' e€ects besides their lipid lowering
properties (for recent reviews see references Rosenson &
Tangney, 1998; Vaughan et al., 2000). In this study we have
investigated the e€ect of di€erent statins on the ®brinolytic
system of human vascular endothelial cells, human vascular
smooth muscle cells and the human hepatoma line HepG2.
There is growing evidence for a role of components of the
®brinolytic system not only in the lysis of blood clots but also in
the development and in the progression of atherosclerosis. High
PAI-1 plasma levels and decreased levels of t-PA are associated
with coronary heart disease and PAI-1 mRNA has been found
Figure 1 (A) E€ect of statins on PAI-1 production in human
umbilical vein smooth muscle cells (HUVSMC). Con¯uent mono-
layers of HUVSMC were incubated for 40 h in the absence or
presence of atorvastatin (open squares), cerivastatin (open circles),
¯uvastatin (open upright triangles), lovastatin (open inverted
triangles), pravastatin (®lled squares) or simvastatin (open diamonds)
at a concentration of 0.1, 0.25, 0.5, 1.0 and 2.5 mM, respectively.
Conditioned media of such treated cells were collected and PAI-1
antigen was determined as described in the Methods section. Values
are given in ng/104 cells/40 h and represent mean values+s.d. of
three independent determinations. Experiments were performed three
times with a representative experiment shown. PAI-1 antigen was
signi®cantly decreased by all statins used except pravastatin at
concentrations 50.25 mM (P50.01); as determined by ANOVA the
concentration response curve for cerivastatin was signi®cantly
di€erent from the concentration response curves obtained for
atorvastatin, ¯uvastatin, lovastatin and simvastatin respectively
(P50.01). Inset (A) Timecourse of the e€ect of ¯uvastatin on PAI-
0.25, 0.5, 1.0 and 2.5 mM, respectively. Conditioned media of such
treated cells were collected and t-PA antigen was determined as
described in the Methods section. Values are given in ng/104 cells/
40 h and represent mean values+s.d. of three independent
determinations. Experiments were performed three times with a
representative experiment shown. t-PA antigen was signi®cantly
increased by all statins used except pravastatin at concentrations
50.5 mM (P50.01); as determined by ANOVA, the concentration
response curve for cerivastatin was signi®cantly di€erent from the
concentration response curves obtained for atorvastatin, ¯uvastatin,
lovastatin and simvastatin respectively (P50.01). Inset (B) Time-
course of the e€ect of ¯uvastatin on t-PA production in human
umbilical vein smooth muscle cells (HUVSMC). Con¯uent mono-
layers of HUVSMC were incubated for 8, 24 or 48 h in the absence
(full diamonds) or presence of 2.5 mM ¯uvastatin (open diamonds).
Conditioned fresh media of such treated cells were collected and t-PA
antigen was determined as described in the Methods section. Values
are given in ng/104 cells and represent mean values+s.d. of three
independent determinations. Experiments were performed three times
with a representative experiment shown. t-PA antigen was signi®-
cantly increased in the presence of ¯uvastatin after 24 h (**P50.01)
and 48 hours (**P50.01) of incubation.
1
production in human umbilical vein smooth muscle cells
(HUVSMC): Con¯uent monolayers of HUVSMC were incubated
for 8, 24 or 48 h in the absence (full diamonds) or presence of 2.5 mM
¯uvastatin (open diamonds). Conditioned media of such treated cells
were collected and PAI-1 antigen was determined as described in the
Methods section. Values are given in ng/104 cells and represent mean
values+s.d. of three independent determinations. Experiments were
performed three times with a representative experiment shown. PAI-1
antigen was signi®cantly decreased in the presence of ¯uvastatin after
24 h (*P50.05) and 48 h (**P50.01) of incubation. (B) E€ect of
statins on t-PA production in human umbilical vein smooth muscle
cells (HUVSMC). Con¯uent monolayers of HUVSMC were
incubated for 40 h in the absence or presence of atorvastatin (open
squares), cerivastatin (open circles), ¯uvastatin (open upright
triangles), lovastatin (open inverted triangles), pravastatin (®lled
squares) or simvastatin (open diamonds) at a concentration of 0.1,
British Journal of Pharmacology vol 135 (1)

288
F. Wiesbauer et al
Statins affect the fibrinolytic system of vascular cells
Table 3 Mevalonate reverses the e€ects of statins on PAI-1 and t-PA production in human umbilical vein smooth muscle cells
(HUVSMC) and in human umbilical vein endothelial cells (HUVEC)
HUVSMC
HUVEC
PAI-1 antigen
No mevalonate Mevalonate No mevalonate Mevalonate No mevalonate Mevalonate No mevalonate Mevalonate
t-PA antigen
PAI-1 antigen
t-PA antigen
Control
310.0+20.0
311.2+43.3 0.40+0.04
0.41+0.06 851.7+87.0
824.3+24.6 0.85+0.03
0.88+0.09
0.91+0.09
0.77+0.03
0.78+0.09
0.77+0.04
0.72+0.03
0.77+0.04
Atorvastatin 194.5+13.3* 322.5+12.2 1.05+0.08**
0.38+0.02 331.7+16.5** 808.7+81.6 1.11+0.23
0.47+0.05 331.9+56.1** 852.3+97.8 0.73+0.02
0.40+0.03 379.9+16.5** 912.0+65.4 1.05+0.15
0.42+0.06 371.7+73.6** 836.2+81.2 1.80+0.12**
0.45+0.05 863.5+88.9
0.49+0.05 363.1+65.1** 831.2+88.5 1.62+0.12**
Cerivastatin
Fluvastatin
Lovastatin
Pravastatin
Simvastatin
170.0+21.1* 302.3+41.2 1.35+0.12**
190.1+12.2* 366.7+41.2 1.16+0.12**
176.6+21.2* 320.0+35.6 1.54+0.15**
322.2+13.4
180.3+21.2* 294.4+28.6 1.57+0.13**
322.5+8.9
0.42+0.02
838.7+40.2 0.75+0.09
Con¯uent monolayers of HUVSMC and HUVEC were incubated for 40 h in the absence or presence of the respective statin at a
concentration of 2.5 mM (HUVSMC) or 5.0 mM (HUVEC) alone or together with mevalonate at a concentration of 100 mM. Conditioned
media of such treated cells were collected and PAI-1 and t-PA antigen was determined as described in the Methods section. Values are
given in ng/104 cells/40 h and represent mean values+s.d. of three independent determinations. **P50.01, *P50.05.
2000; Davidson et al., 1997; Bevilacqua et al., 1997; Leren et al.,
1988; Kostner et al., 1989; Tan et al., 1999; Isaacsohn et al.,
1994). The results of these clinical studies, however, are
inconsistent. E.g. for both ¯uvastatin and lovastatin an increase
and a decrease in PAI-1 levels in plasma was reported in
di€erent clinical investigations (Davidson et al., 1997; Bev-
ilacqua et al., 1997; Leren et al., 1988; Kostner et al., 1989; Tan
et al., 1999; Isaacsohn et al., 1994).
Here we present evidence that all statins tested except
pravastatin concentration dependently decreased PAI-1
production in cultured endothelial cells and smooth muscle
cells isolated from human umbilical vein. Similar results were
obtained when human microvascular endothelial cells and
human coronary artery smooth muscle cells were treated with
statins indicating that the observed e€ect was not speci®c for
a particular type of endothelial cell or smooth muscle cell.
Figure 2 E€ect of statins on PAI-1 production in human umbilical
vein endothelial cells (HUVEC). Con¯uent monolayers of HUVEC
were incubated for 40 h in the absence or presence of atorvastatin
(open squares), cerivastatin (open circles), ¯uvastatin (open upright
Furthermore t-PA production in smooth muscle cells was
also concentration dependently increased by all statins, with
the exception of pravastatin which showed no e€ect. In that
respect it is of interest that in other in vitro studies
pravastatin did not inhibit smooth muscle cell growth and
migration and did not induce apoptosis whereas cerivastatin,
¯uvastatin, simvastatin and lovastatin inhibited proliferation
and migration of these cells and the latter two induced
apoptosis (Sindermann et al., 2000; Stark et al., 1998; Indol®
et al., 2000; Bellosta et al., 1998b). This lack of ecacy of
pravastatin in cell culture studies might be brought about by
its lipophobic properties hindering pravastatin's penetration
of the cell membrane. In addition it should be noted that ±
when comparing the e€ects of the di€erent statins on PAI-1
triangles), lovastatin (open inverted triangles), pravastatin (®lled
squares) or simvastatin (open diamonds) at a concentration of 0.1,
0.25, 0.5, 1.0, 2.5 and 5.0 mM, respectively. Conditioned media of
such treated cells were collected and PAI-1 antigen was determined as
described in the Methods section. Values are given in ng/104 cells/
40 h and represent mean values+s.d. of three independent
determinations. Experiments were performed three times with a
representative experiment shown. PAI-1 antigen was signi®cantly
decreased by all statins used except pravastatin at concentrations
50.25 mM (P50.01); as determined by ANOVA, the concentration
response curve for cerivastatin was signi®cantly di€erent from the
concentration response curves obtained for atorvastatin, ¯uvastatin,
lovastatin and simvastatin respectively (P50.01). Inset to (2)
Timecourse of the e€ect of ¯uvastatin on PAI-1 production in
human umbilical vein endothelial cells (HUVEC). Con¯uent
monolayers of HUVEC were incubated for 8, 24 or 48 h in the
absence (full diamonds) or presence of 5.0 mM ¯uvastatin (open
diamonds). Conditioned media of such treated cells were collected
and PAI-1 antigen was determined as described in the Methods
section. Values are given in ng/104 cells and represent mean
values+s.d. of three independent determinations. Experiments were
performed three times with a representative experiment shown. PAI-1
antigen was signi®cantly decreased in the presence of ¯uvastatin after
24 h (**P50.01) and 48 h (**P50.01) of incubation.
and t-PA production in the cells used in our study
±
atorvastatin, ¯uvastatin, lovastatin and simvastatin showed
similar concentration response curves. The concentration
response curves for cerivastatin in these experiments were
shifted to the left, indicating that signi®cantly lower
concentrations of cerivastatin were necessary to elicit e€ects
on the ®brinolytic system in endothelial cells and smooth
muscle cells comparable to the e€ects induced by the other
statins used. Interestingly the recommended in vivo dosages
for cerivastatin are signi®cantly lower than for the other
HMG CoA reductase inhibitors (Hunninghake, 1998;
in human atherosclerotic lesions (Loskuto€ et al., 1993; Aznar
& Estelles, 1994; Schneiderman et al., 1992). Various in vivo
studies have investigated the e€ects of statins on plasma levels
of PAI-1 (Avellone et al., 1994; Wada et al., 1992; Dangas et al.,
Desager
& Horsmans, 1996; Marais et al., 1997). In
endothelial cells only simvastatin and lovastatin increased t-
PA production whereas the other statins had no e€ect. This is
in agreement with two recent studies that investigated the
British Journal of Pharmacology vol 135 (1)

F. Wiesbauer et al
Statins affect the fibrinolytic system of vascular cells
289
Figure 3 (A) E€ect of statins on PAI-1 and t-PA mRNA expression
in human umbilical vein smooth muscle cells (HUVSMC). Con¯uent
monolayers of HUVSMC were incubated for 16 hours in the absence
or presence of 2.5 mM of the respective statin (lane 1: control, lane 2:
atorvastatin, lane 3: cerivastatin, lane 4: ¯uvastatin, lane 5:
lovastatin, lane 6: simvastatin). mRNA of such treated cells was
prepared and PAI-1, t-PA and GAPDH mRNA was visualized by
Northern blotting as described in the Methods section. Experiments
were performed twice with a representative experiment shown. (B)
E€ect of statins on PAI-1 mRNA expression in human umbilical vein
endothelial cells (HUVEC). Con¯uent monolayers of HUVEC were
incubated for 16 h in the absence or presence of 5.0 mM of the
respective statin (lane 1: control, lane 2: atorvastatin, lane 3:
cerivastatin, lane 4: ¯uvastatin, lane 5: lovastatin, lane 6:
simvastatin). mRNA of such treated cells was prepared and PAI-1
and GAPDH mRNA was visualized by Northern blotting as
described in the Methods section. Experiments were performed twice
with a representative experiment shown.
Figure 4 (A) E€ect of statins on tumor necrosis factor-a (TNF-a)
induced PAI-1 production in human umbilical vein smooth muscle
cells (HUVSMC). Con¯uent monolayers of HUVSMC were
incubated for 40 h in the absence (open bar) or presence of TNF-a
(10.0 nM; full bar), cerivastatin (2.5 mM; hatched bar), or simvastatin
(2.5 mM; hatched bar), respectively, or with the respective statin
together with TNF-a (hatched bars) at the concentrations indicated
above. Conditioned media of such treated cells were collected and
PAI-1 antigen was determined as described in the Methods section.
Values are given in ng/104 cells/40 h and represent mean values+s.d.
of three independent determinations. Experiments were performed
three times with a representative experiment shown. }} Signi®cantly
di€erent from control, P50.01; **signi®cantly di€erent from TNF-a
treated cells, P50.01. (B) E€ect of statins on interleukin-1a (IL-1a)
induced PAI-1 production in human umbilical vein endothelial cells
(HUVEC). Con¯uent monolayers of HUVEC were incubated for
40 h in the absence (open bar) or presence of IL-1a (200 u ml71; full
bar), ¯uvastatin (5.0 mM; hatched bar), lovastatin (5.0 mM; hatched
bar) or simvastatin (5.0 mM; hatched bar), respectively, or with the
respective statin together with IL-1a (hatched bars) at the
concentrations indicated above. Conditioned media of such treated
cells were collected and PAI-1 antigen was determined as described in
the Methods section. Values are given in ng/104 cells/40 h and
represent mean values+s.d. of three independent determinations.
e€ects of these two statins on the ®brinolytic system of
endothelial cells and smooth muscle cells (Essig et al., 1998;
Bourcier & Libby, 2000).
As indicated by the results of a cell viability assay using
lactate dehydrogenase leakage as a marker of cell damage,
the e€ects of the HMG CoA reductase inhibitors on the
®brinolytic system of vascular cells were not due to
cytotoxicity. Lactate dehydrogenase leakage was not a€ected
in endothelial cells by statins at concentrations of 45 mM
whereas in smooth muscle cells a cytotoxic e€ect was
observed at a concentration of 5 mM. No di€erence in lactate
dehydrogenase leakage to control cells was seen in smooth
muscle cells treated with statins at concentrations of
Experiments were performed three times with
a representative
experiment shown. }} Signi®cantly di€erent from control, P50.01;
**signi®cantly di€erent from IL-1a treated cells, P50.01.
42.5 mM. The e€ects of statins on PAI-1 and t-PA
production could be completely reversed by mevalonate
indicating that these e€ects were brought about by inhibition
British Journal of Pharmacology vol 135 (1)

290
F. Wiesbauer et al
Statins affect the fibrinolytic system of vascular cells
of the mevalonic acid pathway. This is in agreement with
several other cell culture studies (Sindermann et al., 2000;
Laufs et al., 1997; Bellosta et al., 1998a; Essig et al., 1998;
Bourcier & Libby, 2000).
The e€ects of statins on components of the ®brinolytic
system on the level of proteins were also re¯ected on the level
of speci®c mRNA expression. As evidenced by Northern
blotting, statin treatment decreased the steady state level of
PAI-1 speci®c mRNA in endothelial cells and smooth muscle
cells whereas levels of t-PA speci®c mRNA were increased in
smooth muscle cells after incubation with HMG CoA
reductase inhibitors.
Furthermore we could show that statins signi®cantly
reduced the IL-1a and TNF-a induced increase in PAI-1 in
vascular cells. Thus we present evidence that HMG CoA
reductase inhibitors might ameliorate the response of vascular
cells towards activation by in¯ammatory cytokines. Recently
it was shown that ¯uvastatin and atorvastatin modestly
inhibited TNF-a induced PAI-1 synthesis in endothelial cells,
whereas simvastatin was shown to inhibit the platelet derived
growth factor (PDGF)- and transforming growth factor-b
(TGF-b) stimulated expression of PAI-1 in vascular cells
(Lopez et al., 2000; Bourcier & Libby, 2000).
produce PAI-1 in an up to 100-fold excess over t-PA leading
to complex formation between t-PA and PAI-1 and total
inhibition of the plasminogen activator by its inhibitor. Thus
no t-PA activity is detectable in supernatants from cultured
human endothelial cells and smooth muscle cells (Bartha et
al., 1988; Wojta et al., 1991; 1993). In contrast, in freshly
isolated human endothelial cells and in resting murine
endothelial cells in vivo PAI-1 is hardly detectable whereas
t-PA is present throughout the vasculature (van den Berg et
al., 1988; Fearns et al., 1995; Kristensen et al., 1984). If the
e€ects of statins on PAI-1 and t-PA expression in vascular
cells in vitro are also operative in vivo, one could therefore
hypothesize that at sites of vascular lesions a reduced
expression of PAI-1 and an increased expression of t-PA by
endothelial cells and smooth muscle cells would increase the
®brinolytic capacity locally thereby favouring thrombolysis
and reducing ®brin formation after plaque rupture. It should
be emphasised that we have shown in this study that statins
exert their downregulating e€ect on PAI-1 production also in
the presence of in¯ammatory cytokines, which are thought to
be present at such sites of plaque rupture and which in the in
vivo setting result in strong induction of PAI-1 expression in
the endothelium (Loskuto€ et al., 1993). Again, if statins
downregulate PAI-1 production in vascular cells activated by
in¯ammatory mediators to basal levels also in vivo, one could
speculate that the ®brinolytic potential of these cells would be
increased and local ®brinolysis would be favoured. On the
In contrast to the e€ects seen in vascular cells, statins at
5.0 mM did not regulate PAI-1 production in the human
hepatoma cell line HepG2 which has been extensively used as
a
model to study the regulation of PAI-1 in human
hepatocytes (Wojta et al., 1994; Healy & Gelehrter, 1994;
Arts et al., 1999). To account for possible metabolization of
the statins by HepG2 cells, the cells were incubated with the
respective HMG CoA reductase inhibitor at a concentration
of 100.0 mM. Even at this high concentration no e€ect on
PAI-1 production in these cells was seen. Cell viability of
HepG2 cells was not a€ected by statins used at concentra-
tions of 4100.0 mM. Thus one could speculate that the e€ects
of statins on the ®brinolytic system might be tissue speci®c.
In general, caution should be used in extrapolating results
of experimental studies to the clinical setting. The concentra-
tions of statins used in this in vitro study are higher than
observed plasma concentrations in patients treated with these
HMG CoA reductase inhibitors (Desager & Horsmans,
1996). They are, however, in the same range as concentra-
tions of statins used in numerous other tissue culture studies
(Sindermann et al., 2000; Indol® et al., 2000; Laufs et al.,
1997; Hernandez-Perera et al., 1998; Romano et al., 2000;
Bellosta et al., 1998a; Ferro et al., 2000; Essig et al., 1998;
Bourcier & Libby, 2000). Furthermore one could speculate
that the local concentration in tissue in the in vivo situation
might depend on the exposure time of the tissue to the HMG
CoA reductase inhibitors. Considering the fact that in most
clinical studies patients are treated with statins over a period
of days or even weeks, these exposure times are much longer
as compared to the in vitro setting where cells are treated with
these drugs only for hours. Given the di€erent response of
vascular cells and HepG2 cells towards statin treatment,
further studies are needed to investigate a possible tissue
speci®city of the described e€ect of statins on components of
the ®brinolytic system.
other hand a decrease in PAI-1 might also favour
extracellular matrix degradation by proteases thereby leading
to plaque instability. Di€erent studies investigating the e€ect
of statins on PAI-1 plasma levels in patients have produced
inconsistent results (Avellone et al., 1994; Wada et al., 1992;
Dangas et al., 2000; Davidson et al., 1997; Bevilacqua et al.,
1997; Leren et al., 1988; Kostner et al., 1989; Tan et al., 1999;
Isaacsohn et al., 1994). The contribution of endothelial cells
under normal conditions to the amount of PAI-1 present in
plasma remains to be de®ned. Freshly isolated human
endothelial cells produce only little PAI-1 and no PAI-1
mRNA could be detected in normal murine endothelium in
vivo (van den Berg et al., 1988; Fearns et al., 1995). PAI-1 has
been detected in human endothelium in vivo but these
®ndings should be interpreted with some caution since most
of these tissues have been obtained under stressed conditions
(e.g. after trauma or major surgery) and PAI-1 is an acute
phase protein (Healy & Gelehrter, 1994; Lupu et al., 1993;
Chomiki et al., 1994). Other possible sources of plasma PAI-1
are platelets, liver cells and adipocytes (for a review see
Loskuto€ & Samad, 1998). Thus
contribution of endothelial cells to plasma PAI-1
±
given the doubtful
we
±
hypothesize that statins might not in¯uence systemic plasma
PAI-1 levels by decreasing its expression in vascular
endothelial cells, but rather exert their clinically proven
bene®cial e€ects by shifting the balance locally at the site of
vascular lesions towards increased ®brinolysis thereby
reducing the risk of thrombus formation at such sites.
This work was supported by the Ludwig Boltzmann Foundation
for Cardiovascular Research and by the Association for the
Promotion of Research in Arteriosclerosis, Thrombosis and
Vascular Biology. The artwork of Thomas Nardelli is gratefully
acknowledged.
We present evidence that in vascular cells all statins tested
except pravastatin reduced PAI-1 and increased t-PA
expression. It should be noted, however, that under culture
conditions human endothelial cells and smooth muscle cells
British Journal of Pharmacology vol 135 (1)

F. Wiesbauer et al
Statins affect the fibrinolytic system of vascular cells
291
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(Received June 11, 2001
Revised September 12, 2001
Accepted October 24, 2001)
British Journal of Pharmacology vol 135 (1)