(Stroke. 1999;30:651-655.)
© 1999 American Heart Association, Inc.
Nicotine Increases Plasminogen Activator Inhibitor-1 Production by Human Brain Endothelial Cells via Protein Kinase CAssociated Pathway
Raphael Zidovetzki, PhD;
Peijia Chen, MD;
Mark Fisher, MD
Florence M. Hofman, PhD
From the Departments of Biology and Neuroscience, University of
California, Riverside, Calif (R.Z.), and the Departments of Pathology (P.C.,
F.M.H.) and Neurology (M.F.), University of Southern California School of
Medicine, Los Angeles, Calif.
Correspondence to Dr Florence Hofman, Department of Pathology, University of Southern California School of Medicine, 2011 Zonal Ave, Los Angeles, CA 90033. E-mail hofman{at}hsc.usc.edu
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Abstract
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Background and PurposeSmoking
both increases stroke risk
and reduces the risk of
thrombolysis-associated intracerebral
hemorrhage.
Plasminogen activator
inhibitor-1 (PAI-1) is a major regulator
of
fibrinolysis; elevation of PAI-1 is associated with an
increased
risk of thrombotic disorders. We studied the effect of
nicotine,
an important constituent of cigarette smoke, on PAI-1
production
by human brain endothelial cells.
MethodsAdult human central nervous system
endothelial cells (CNS-EC) were used for tissue culture
experiments. We analyzed culture supernatant for PAI-1 protein
and measured PAI-1 mRNA (by Northern blot analysis) and protein
kinase C (PK-C) activity.
ResultsNicotine at 100 nmol/L increased PAI-1 protein
production and mRNA expression by CNS-EC. After 72 hours of
exposure to nicotine, the concentration of secreted PAI-1 in the cell
supernatant was increased 1.90±0.2 fold compared with untreated cells.
PAI-1 mRNA also increased approximately twofold. Inhibition of PK-C
completely abolished this effect. Nicotine had no effect on the
concentration of tissue plasminogen
activator.
ConclusionsNicotine increases brain endothelial
cell PAI-1 mRNA expression and protein production via
PK-Cdependent pathway. These findings provide new insights into why
smoking may be associated with predisposition to thrombosis and
inversely associated with intracerebral
hemorrhage after therapeutic tissue plasminogen
activator therapy.
Key Words: cerebral ischemia endothelium nicotine plasminogen activator inhibitor 1 protein kinase C
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Introduction
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Cigarette smoking is an important stroke risk factor:
smoking
increases the risk of stroke by approximately
50%.
1 Stroke
risk increases with increasing cigarette
consumption.
2 The
mechanism of the increased stroke risk
has been attributed to
both procoagulant and atherogenic effects of
smoking.
3 4 Analysis
of the landmark National
Institutes of Health (NIH) tissue plasminogen
activator
(tPA) trial has shown that cigarette smoking
protects tPA-treated
patients from cerebral
hemorrhage.
5 6 The prevalence of
intracerebral
hemorrhage among smokers
receiving tPA was 4%, as compared with
13% of the patients who did
not smoke. This observation is especially
relevant because the
therapeutic benefits of tPA in acute stroke
are partially outweighed by
a more than a sixfold increased
risk of symptomatic
intracerebral hemorrhage.
5 6
The mechanism of this dual role for smoking (increasing the risk for
stroke while protecting from hemorrhage in the setting of acute
stroke therapy) has been unclear. One possible mechanism involves
smoking modulation of fibrinolysis, which requires the
conversion of plasminogen into plasmin by tPA. tPA is
rapidly inactivated by plasminogen activator inhibitor-1 (PAI-1),
the predominant plasminogen activator
inhibitor in human plasma.7 8 9 PAI-1 is
synthesized by endothelial cells (ECs), human
hepatocytes, granulosa, and vascular smooth muscle cells
and comprises up to 12.5% of the protein secreted by ECs in
culture.9 10 Increased plasma PAI-1 is found in patients
with ischemic stroke,11 in survivors of myocardial
infarction,12 13 and in other thrombotic
disorders.14 15 16 17
In the present study we investigated the relationship between PAI-1
and nicotine, an important component of cigarette smoke. This is
particularly relevant because nicotine has previously been shown to
alter rat brain capillary endothelial
fibrinolysis and to increase infarct size in a middle
cerebral artery occlusion model.18 Our results here
demonstrate that concentrations of nicotine achieved in the
plasma/serum of smokers increase PAI-1 production by central
nervous system (CNS)EC. The protein kinase C (PK-C) pathway was
chosen for investigation because it is a common intracellular signal
transduction mechanism and therefore would be relevant to understanding
the regulation of PAI-1 production.
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Materials and Methods
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Cell Culture
CNS-ECs were derived from human brain as previously described
in
detail.
19 Cells were cultured in RPMI-1640 medium (GIBCO
Labs)
supplemented with 100 ng/mL endothelial
cell growth factor Endogro
(VECTEC), 2 mmol/L
L-glutamine, 10 mmol/L Hepes, 24 mmol/L sodium
bicarbonate,
300 USP U of heparin, 1%
penicillin
/streptomycin, and 10% fetal
calf serum. We used
subconfluent preparations of cells up until
passage 4 to 5 only; and
Endogro-free medium was used beginning
24 hours before the experiment.
The purity of CNS-EC (>95%)
was confirmed by immunocytochemical
staining for factor VIII,
glial fibrillary acidic protein, and the
macrophage maker CD11b,
as previously described.
19
The PK-C inhibitor GF-109203-X (GF)
(Calbiochem) was added
to the cells 30 minutes before nicotine
treatment. GF at 1
µmol/L was used in inhibition experiments;
this dose completely
inhibited PK-C in CNS-EC without affecting
cell viability
(authors' unpublished observations). Nicotine
sulfate (Sigma)
was used at 100 nmol/L, on the basis of titration
experiments and
physiological range.
PK-C Assay
ECs were treated as described in the previous sections. The PK-C
assays were performed using PK-C assay kits obtained from GIBCO.
Briefly, the experimental treatments were terminated by replacing the
medium with the cell extraction buffer (20 mmol/L TRIS, 0.5
mmol/L EDTA, 0.5 mmol/L EGTA, 25 µg/mL each aprotinin and
leupeptin, pH 7.5) at room temperature. The cells were then
homogenized with a precooled Dounce
homogenizer, and the cytosol and membrane fractions
were separated by centrifugation (10 000g,
30 minutes), at 4°C. The supernatants were collected as the cytosol
fraction. The pellets were resuspended in 0.5 mL extraction buffer with
the detergent (1% NP-40). Membrane fractions were obtained by
centrifugation (10 000g, 10 minutes, 4°C)
and the supernatant was collected. The determinations of PK-C activity
in the cytosol and membrane fractions were performed according to the
manufacturer's instructions. The specific PK-C substrate was a
synthetic peptide from myelin basic protein (amino acids 4 to 14) with
an acetylated N-terminal glutamine, and the specific
inhibitor was a peptide (amino acids 19 to 36) derived from
the same protein that binds to the "pseudosubstrate" region of the
regulatory domain. The specific PK-C activity was determined as the
difference between phosphorylation of the
PK-Cspecific substrate in the absence or presence of the specific
PK-C inhibitor. The data are presented as ratios of
the membrane and the total (cytosol+membrane) PK-C activities.
PAI-1 Production
PAI-1 production was evaluated using the commercially
available ELISA kit (American Diagnostics Inc). Briefly,
cells were grown in culture to 70% to 80% confluence in 10% fetal
calf serum. Culture supernatants (100 µL) were removed after 24, 48,
72, and 96 hours and evaluated for PAI-1 content. The ELISA determined
the amounts of bound and free PAI-1 present. Each data point was
generated by samples run in triplicate; and each experiment was
repeated at least 3 times.
RNA Analysis
RNA from 2 to 3x106 cultured cells was
isolated 24 hours after treatment and prepared according to a
modification of the acid phenol method using the Trisol reagent (Life
Technologies) as specified by the manufacturer. Twelve micrograms of
total RNA was denatured and fractionated on a 1.2% agarose gel
containing formaldehyde, then transferred to
Hybond-N+ nylon membrane (Amersham), and
hybridized with [32P]dCTP Klenow-labeled
random-primed probes (Boehringer Mannheim) according to
manufacturer's instructions. Prehybridization was performed in Quikhyb
hybridization solution (Stratagene) for 15 minutes at 68°C.
Subsequently the labeled probe plus 1 mg salmon sperm DNA was added to
the hybridization solution mixture and incubated for 1 hour at 68°C.
The hybridized membrane was washed twice with 2x standard saline
citrate, 0.1% sodium dodecyl sulfate at 37°C, then washed
with 0.1x standard saline citrate, 0.1% sodium dodecyl
sulfate buffer for 15 minutes at 60°C. Membranes were exposed to
Hyperfilm-MP (Amersham) for at least 24 hours at -70°C. The human
PAI-1 probe used in hybridization, obtained from plasmid designated as
pPAI-1 1.3, is 1253 bp in length20 and binds to the
full-length PAI-1 mRNA (3.0 kb). Densities of hybridized bands were
determined by scanning and analyzed using the NIH Image
software program. Blots were rehybridized with labeled polymerase chain
reaction products of glyceraldehyde-3 phosphate
dehydrogenase (GAPDH; Stratagene), the housekeeping gene, to control
for total amount of RNA present in the cells and precision of RNA
loading.
Statistics
All experiments were performed 3 times unless otherwise stated.
The error bars on all figures correspond to SEM. Statistical
comparisons between groups were performed using unpaired Student's
t tests or 2-way ANOVA. All data presented as
significant in this study have the value of P<0.02, unless
otherwise indicated.
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Results
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We investigated the effects of physiological
concentrations
of nicotine on PAI-1 production by human
brainderived
endothelial cells. CNS-ECs were cultured
in the absence or presence
of nicotine (100 nmol/L) for 96 hours.
Aliquots of supernatant
were removed after 24, 48, 72 and 96 hours and
analyzed for
secreted PAI-1 protein using the ELISA technique.
The results
of 4 experiments (each in triplicate) on different CNS-EC
primary
cell cultures demonstrated a significant increase in PAI-1
production
following nicotine treatment after 72 hours with a
continued
increase through 96 hours of culture. Primary cultures
typically
exhibit great variability in baseline levels of PAI-1
production,
ranging from 9.6 to 33.4 µg/mL per
10
6 cells (27.1±2.9;
SEM), and after 72 hours of
nicotine treatment, ranging from
31.8 to 75.2 µg/mL per
10
6 cells (53.8±5.8; SEM).
The results of
nicotine treatment were therefore assessed in
terms of relative PAI-1
production for each treated culture
as compared with its
respective control. Nicotine treatment
increased relative PAI-1
production by 1.9±0.2 fold (
P<0.02).
To determine
whether nicotine affects PAI-1 mRNA expression,
CNS-ECs were exposed to
nicotine for 24 hours, and RNA was isolated
and analyzed for
PAI-1 mRNA. The results (Figure 1A

, 1B

) show
that PAI-1 mRNA was increased approximately twofold in
the presence
of nicotine. The data presented are
representative of 3 experiments
performed. Incubation
with nicotine for 1, 4, or 6 hours showed
no significant increase in
PAI-1 mRNA above control values (data
not shown). Thus, nicotine
increases PAI-1 production on mRNA
and protein levels. In
contrast, incubation of the CNS-ECs with
nicotine (100 nmol/L) for 72
hours did not significantly affect
tPA production
(14.4±1.7 ng/mL · 10
6 cells for
the
control versus 15.5±1.6 ng/mL · 10
6 cells
for
nicotine-treated cells).

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Figure 1. Effect of nicotine on mRNA expression by CNS-EC.
A, CNS-EC cultures were exposed to media alone, nicotine (100 nmol/L),
GF (1 µmol/L) alone, or GF and nicotine for 24 hours. Northern
blot analyses for human PAI-1 and GAPDH are presented.
B, ratios of scanned density values of PAI-1 to GAPDH are
presented.
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We next determined whether the PK-Cassociated intracellular signal
transduction pathway is involved in a nicotine-induced increase in
PAI-1 production. The effect of nicotine on PK-C activity was
measured as a function of time. Figure 2
shows that nicotine (100 nmol/L) indeed caused an increase in PK-C
activity in ECs. The increase in PK-C activity was significant within
30 seconds, attained maximum levels at 2 minutes, and sharply declined
thereafter, reaching control levels at 30 minutes (Figure 2
). To
determine the significance of the nicotine-induced PK-C activation in
the observed increase in PAI-1 production, we measured the
effect of nicotine in the presence of the PK-C inhibitor
GF. Figure 1
demonstrates that in the presence of GF, nicotine
has no effect on PAI-1 mRNA levels. Similar results were obtained with
another PK-C inhibitor calphostin C (data not shown).
Furthermore, Figure 3
shows that GF
completely inhibited nicotine-induced PAI-1 protein production,
demonstrating that in CNS-ECs PAI-1 mRNA expression and protein
production are dependent on the activation of PK-C.

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Figure 2. Effect of nicotine on PK-C activity of CNS-EC.
CNS-EC were exposed to nicotine (100 nmol/L) and PK-C activity was
measured in cytosol and membrane fractions as a function of the
duration of nicotine treatment.
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Figure 3. Effect of PK-C inhibitor GF on
nicotine-induced PAI-1 production. Cultures of CNS-EC were
exposed to nicotine (100 nmol/L), GF (1 µmol/L), or both
reagents for 72 hours. Subsequently supernatants were tested for PAI-1
production (per 106 cells) using the ELISA
technique. Nicotine significantly increased PAI-1 production
(P<0.02). In the presence of GF, there was no
significant difference between nicotine-treated and control
cells.
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Discussion
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We have shown that nicotine increases PAI-1 production by
human
CNS-ECs. After 72 hours of incubation, PAI-1 protein
production
increased twofold; this paralleled the increase
in PAI-1 mRNA
expression. These effects were completely abolished by
inhibition
of PK-C, indicating that nicotine induces increased PAI-1
production
in CNS-ECs via a PK-Cdependent pathway.
We observed the effects of nicotine at 100 nmol/L, within the
concentration range of nicotine achieved in the serum of
smokers.21 22 Previous studies of the biological effects
of nicotine in various vascular systems have shown that mice
chronically exposed to nicotine exhibit subendothelial
edema, endothelial cell swelling, cytoplasmic
vacuolation, and mitochondrial swelling.23 Exposure of
bovine EC to nicotine caused giant cell formation, ruffled membrane,
and extensive cellular vacuolation.24 25 Other studies
found no significant effects of nicotine on EC at the submicromolar
concentrations. No change of functions such as growth properties,
morphology, total protein and collagen synthesis and turnover, lactate
production, DNA synthesis, and
platelet-subendothelial interaction/adhesion was
observed in bovine aortic ECs with up to 10-4
mol/L nicotine in 1 to 4 days.26 Thus, there is no
consensus in the literature regarding the effects of nicotine on
ECs.
This uncertainty may be related to the use of different nicotine
concentrations and duration of the treatment and may depend on the
target tissue. Specifically, there are significant
physiological differences between micro- and
macrovascular ECs. Several growth factors are specific for one or the
other type.27 28 29 Coagulation factors are expressed
differently between large vessels and capillaries. Thrombin stimulates
ECs from large vessels but not from the capillaries.30
Elevation of glucose resulted in decreased PAI-1 mRNA in brain ECs but
not in human umbilical vein ECs (HUVECs) or bovine aortic
ECs.31 The PK-C activator PMA suppresses the
basic fibroblast growth factorinduced proliferation of capillary ECs
but has no effect on aortic ECs.32 Also, vanadate
treatment leads to an inhibition of protein tyrosine kinase activity in
the aortic ECs but not in the capillary ECs.33 Thus, these
ECs differ not only in the array of growth factor receptors present
on their surfaces but also in their intracellular regulatory
mechanisms. For these reasons we have used human adult CNSderived
ECs, emphasizing the relevance to stroke. Our results, which associated
increased PAI-1 production with activation of PK-C, agree with
other studies in which PK-C activation was shown to increase PAI-1
production in various ECs, including
HUVECs,9 34 35 36 bovine CNS-ECs,31 and bovine
aortic ECs.31 37 Furthermore, our data are
consistent with clinical findings showing increased plasma
levels of PAI-1 in smokers.38 39
There is a complex relationship between smoking and hemorrhagic
transformation after tPA treatment for stroke. Hemorrhagic
transformation is related to disruption of the microvascular basal
lamina after cerebral ischemia,40 and it is
unclear how that process is modified by therapeutic
thrombolysis and smoking. However, it is known that
smokers have increased plasma levels of PAI-1,38 and there
is substantial evidence emphasizing the importance of PAI-1 in
thrombolysis after acute myocardial infarction.
Measurements of PAI-1 predict outcome in these patients: high baseline
PAI-1 levels are associated with occluded coronary arteries 90
minutes after treatment.41 Moreover, neutralizing
antibodies to PAI-1 produce enhanced efficacy of therapeutic
thrombolysis in an experimental model of
coronary artery thrombosis.42 In the setting of
acute arterial thrombosis, endothelial- and
platelet-derived PAI-1 bind to fibrin strands and
inactivate tPA.42 43 44
It should be emphasized that nicotine is only one component of
cigarette smoke. It is thought that much of the toxic effects of
smoking are due to a variety of gas-phase constituents and to tar
rather than nicotine.45 It is unclear to what extent
nicotine contributes to hemostatic alterations in humans. Subjects
receiving transdermal or oral nicotine have plasma levels of
platelet activation products
(ß-thromboglobulin and platelet factor 4) and
von Willebrand factor that tend to be intermediate between
smokers and control subjects,46 47 suggesting possible
synergistic effects among the different cigarette components. With the
use of our in vitro culture system, the effects of nicotine alone on
brain-derived ECs are evident.
In summary, our results demonstrate that nicotine induces PAI-1
production in CNS-ECs and that this process is mediated through
the PK-Cdependent pathway. Enhanced production of PAI-1 may
serve to increase stroke risk by increasing predisposition to
thrombosis and to reduce hemorrhagic risk by inactivating therapeutic
tPA. Thus, clarification of the mechanism of PAI-1 regulation by
nicotine may allow therapeutic modification of both harmful and
potentially useful effects.
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Acknowledgments
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This work was supported by NIH grant PO1-NS31945.
Received July 20, 1998;
revision received December 7, 1998;
accepted December 8, 1998.
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Wagner OF, de Vries C, Hohmann C, Veerman H, Pannekoek
H. Interaction between plasminogen activator
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Benowitz NL. The role of nicotine in smoking-related
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Benowitz NL, Fitzgerald GA, Wilson M. Zhang Q.
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Editorial Comment
Frank M. Faraci, PhD, Guest Editor
Department
of Internal Medicine,
Cardiovascular Center,
University of Iowa College of Medicine,
Iowa City, Iowa
 |
Introduction
|
|---|
Nicotine is known to have several effects on blood vessels,
including
vasoconstriction, increased production of superoxide
anion,
and impairment of endothelium-dependent
relaxation.
1 2 The
preceding article summarizes a study
that examined effects of
nicotine on production of
plasminogen activator inhibitor-1
(PAI-1)
by cerebral endothelium. PAI-1 is a major
regulator of fibrinolysis.
Increased levels of PAI-1
are present in atherosclerotic lesions,
3 and elevated
plasma levels of PAI-1 are associated with increased
risk for
thrombotic disorders.
These are several findings in this study. First,
physiologically relevant concentrations of
nicotine increased endothelial production of
PAI-1 by approximately 2-fold. Second, a molecular analysis
indicated that increased expression of PAI-1 in response to nicotine
occurred at the mRNA and protein levels. Third, biochemical and
pharmacological evidence suggests that the mechanism responsible for
increased production of PAI-1 in response to nicotine involves
activation of protein kinase C. These new findings, along with other
known effects of nicotine on blood vessels, support the concept that
this important component of cigarette smoke may contribute to vascular
dysfunction and a predisposition to thrombosis.
Received July 20, 1998;
revision received December 7, 1998;
accepted December 8, 1998.
 |
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