Thrombosis |
From the Departments of Biology and Neuroscience (R.Z.), University of California, Riverside; and Departments of Pathology (J.-L.W., P.C., F.M.H.) and Neurology (J.A.K., M.F.), University of Southern California School of Medicine, Los Angeles.
Correspondence to Florence M. Hofman, PhD, USC School of Medicine, HMR 312, 2011 Zonal Ave, Los Angeles, CA 90033.
| Abstract |
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(TNF-
) acted synergistically to increase
PAI-1 production. ET-1 activated protein kinase C and
cAMP-dependent protein kinase pathways within 3 to 5 minutes of
treatment, with the peak at 10 minutes. Activation of protein kinase C
by phorbol-12-myristate-13-acetate (PMA) resulted in increased
PAI-1 production, whereas activation of the cAMP-dependent
protein kinase by forskolin or dibutyryl cAMP (dBu-cAMP) significantly
decreased PAI-1 production. However, simultaneous
activation of protein kinase C by PMA and cAMP-dependent protein kinase
by dBu-cAMP only slightly attenuated PMA-induced PAI-1 increase.
Inhibition of protein kinase C by GF-109213X abolished the
effects of ET-1. These results demonstrate that ET-1 and TNF-
function synergistically to induce procoagulant activity of brain
endothelial cells in a process that involves a protein
kinase C-dependent pathway.
Key Words: endothelin plasminogen activator inhibitor 1 brain endothelium protein kinase C cAMP
| Introduction |
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Clot formation and lysis is regulated, in part, by the potent
proteolytic enzyme plasmin (see review in Reference 2323 ). Inactive
plasminogen is cleaved and converted to active plasmin by
the enzyme tissue plasminogen activator (t-PA.)
The t-PA is rapidly inactivated by plasminogen
activator inhibitor (PAI).24 Thus,
net fibrinolytic activity in vivo is the result of the balance between
the levels of t-PA and PAI.25 26 Of several PAI types,
PAI-1 appears to be the most important in plasma.27 Serum
PAI-1 levels have been found to be elevated in myocardial
infarction,28 29 30 and are associated with an increased
risk for thrombotic disease.31 32 EC produce t-PA, PAI-1,
and PAI-2,33 34 which are regulated by factors such as
thrombin, histamine, endotoxin, and cytokines, eg,
interleukin-1ß (IL-1ß) and tumor necrosis factor-
(TNF-
).35 36 37 38 39 These proinflammatory cytokines
act directly on EC by stimulating procoagulation
activity.40
In this study, human brain-derived EC were treated with ET-1 and
examined for PAI-1 production. The data here demonstrate that
ET-1 upregulates both PAI-1 mRNA and protein levels, as well as PAI-1
activity; and this PAI-1 induction is mediated by a protein kinase C
(PK-C)-dependent pathway. Furthermore, TNF-
functions
synergistically with ET-1 to increase PAI-1 production.
| Methods |
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was purchased from Boehringer-Mannheim. ET-1 was
obtained from Peninsula Laboratory.
Cell Culture
CNS EC were derived from human brain as previously
described.41 Briefly, brain tissue, obtained after
surgery, was digested with a series of
collagenase/dispase treatments; cells were isolated
on the basis of acetylated-LDL binding and FACS cell sorting.
Purity of CNS EC was confirmed by immunocytochemical staining for
factor VIII, glial fibrillary acidic protein, and the
macrophage marker CD11b, as previously
described41 ; recovery routinely was >95% of CNS EC.
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 U of heparin, 1%
penicillin/streptomycin, and 10% FCS. Only cells from passage 4
to 5 were used in the described experiments; Endogro-free medium was
used 24 hours before the initiation of experiments. The
inhibitor GF (1 µmol/L) was added to cultures 30
minutes before ET-1 treatment.
PAI-1 Production
PAI-1 production was evaluated using the commercially
available ELISA kit (American Diagnostics Corp). Briefly,
cells were grown in culture to 60% to 70% confluence in 10% FCS.
Culture supernatants (100 µL) were removed after 24, 48, and 72 hours
and evaluated for PAI-1 content. The ELISA determined the total amount
of bound and free PAI-1 present. The data for PAI-1 and t-PA are
represented as the ratios of the concentrations of PAI-1 or
t-PA protein derived from the supernatants of stimulated cell cultures
compared with control cultures. The average control value for PAI-1 was
16.3±3.0 µg/mL per 106 cells; the average
control value for t-PA was 43.5±2.3 ng/mL per
106 cells. Each experiment was performed 3 times,
unless otherwise stated.
RNase Protection Assay
The radioactively labeled RNA antisense probe for PAI-1 was
prepared using PCR; the ß-actin template was provided by Ambion.
Using the In Vitro Transcription Kit (Ambion), 6 µL of
32P CTP (800 Ci/mmol, 10 mCi/mL; NEN Research) and 2 µL
each of 10x transcription buffer, T7 polymerase, DTT, and RNAsin were
added as suggested by the manufacturer. The reaction was
terminated by adding 1 µL RNase-free DNase for 1 hour at 37°C. The
probes were then extracted using Tris-saturated
phenol:cholorform:isoamyl alchol (25:24:1) (GIBCO BRL) and
chloroform:isoamyl alcohol sequentially, and then precipitated with
ethanol. The radiolabeled RNA pellet was air dried and solubilized with
hybridization buffer. RNA was isolated from 2 to
3x106 cells per experimental group and prepared
according to a modification of the acid phenol method using the Trizol
reagent (Life Technologies) as specified by the manufacturer. Total RNA
(10 µg) together with 6x105 cpm of probe was
heat denatured at 90°C and then hybridized overnight at 56°C.
Subsequently, the samples were treated with the RNase cocktail,
followed by proteinase K cocktails, then precipitated using ammonium
acetate and ethanol. Air-dried samples were solubilized in 1x loading
buffer, denatured at 90°C for 3 minutes, and then placed on ice. The
protected fragments were resolved in 5% acrylamide 8 mol/L
urea gel; the gel was dried and exposed to Hyper film (Amersham Life
Science) at -70°C. The protected bands were observed for PAI-1 (614
bp) and ß-actin (320 bp). The differences among the groups were
calculated as the ratio of the spectrophotometric densities of the
experimental to control groups. The manufacturer's recommended yeast
tRNA negative control and a positive control were included in every
RNase protection assay (RPA) experiment.
PAI-1 Activity Assay
The PAI-1 activity assay was performed using Pharmacia Coates
PAI kit. A fixed amount of t-PA was added in excess to the conditioned
media of the treated samples. A portion of t-PA forms an inactive
complex with the PAI-1; subsequently, plasminogen was
activated to plasmin by residual t-PA in the presence of the
stimulator. The amount of plasmin formed is directly proportional to
the PAI-1 activity in the conditioned medium. In the presence of
chromogenic substrate, the color change was read at 405
nm.
cAMP Assay
The cAMP assays were performed using a kit (DuPont), according
to the manufacturer's instructions. Briefly, 2 to
3x105 EC were plated in media containing 10%
FCS with no Endogro in 60-mm culture dishes 1 to 2 days before the
experiment. The cells were grown to near (80% to 90%) confluence; 2
hours before initiating the experiment the growth medium was replaced
with medium containing 1% FCS. The cells were treated with different
agents for the specified time; the incubation was terminated by
replacing the medium with 1 mL of 6% (wt/vol) trichloroacetic acid.
The cells were then collected, homogenized with glass
dounce homogenizer, and placed on ice for 30 minutes.
The cell suspensions were centrifuged at 1500g for
15 minutes at 4°C. The supernatants were collected, and the
trichloroacetic acid was removed by washing with water-saturated ether
3 times. Samples were lyophilized and redissolved in cAMP assay buffer
(1 mol/L sodium acetate, 0.6% EGTA, 0.2% sodium azide). A portion of
the supernatant (100 µL) was acetylated to increase
sensitivity. The cAMP content of the cells was subsequently measured by
RIA (Rianen Assay System). The standard curve was obtained with
unlabeled standard cAMP antigen (range, 0.1 to 4 pmol/mL) and a fixed
amount of the [125I]-labeled antigen reacted
with a constant and limiting amount of antibody; a decreasing amount of
the labeled antigen is bound to the antibody as the amount of unlabeled
standard antigen is increased. These data were used to construct a
standard curve from which the values in the samples were determined by
interpolation. The data are expressed as mean±SD. All experiments
presented here were performed at least 3 times unless otherwise
stated.
PK-A Assay
EC were treated as described for the cAMP assays. The treatments
were terminated by rinsing the cells with Dulbecco's PBS and scraping
them off with 0.5 mL of extraction buffer (5 mmol/L EDTA, 50
mmol/L Tris, pH 7.5.) The cell suspensions were then
homogenized and sedimented (10 000g, 10
minutes). The supernatants were collected for PK-A activity
determination. The reaction was initiated by adding 10 µL of the
lysates to 20 µL of reaction buffer; subsequently 10 µL of
[32P]ATP was added. The PK-A activity was
determined as the difference between phosphorylation of
a PK-Aspecific substrate (Leu-Arg-Arg-Ala-Ser-Leu-Gly, kemptide) with
or without a specific peptide PK-A inhibitor, which has an
alanine to serine replacement in the consensus sequence
Xaa-Arg-Arg-Xaa-Ser-Xaa and binds to the pseudosubstrate region of the
regulatory domain of PK-A. The reaction was allowed to proceed at
30°C for 15 minutes and was terminated by removing a 20-µL aliquot
to a phosphocellulose filter. Free [32P]ATP was
removed by washing the filter twice with both 1% phosphoric acid and
water. Substrate phosphorylation was quantified by
measuring the radioactivity on the phosphocellulose filters in
scintillation fluid (Beta-fluor). All experiments were repeated at
least 3 times and the data presented as mean±SD.
PK-C Assay
EC 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, and 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, 4°C). The supernatants were collected as the cytosol
fraction. The pellets were resuspended in 0.5 mL of extraction buffer
with the detergent (1% NP-40). Membrane fractions were obtained by
centrifugation (10 000g, 10 minutes, 4°C)
and collecting the supernatants. PK-C in both membrane and cytosol
fractions was partially purified by ion exchange
chromatography (DE-52 cellulose column). 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 specific PK-C 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.
| Results |
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, tested at an optimal
concentration (10 ng/mL) alone or in the presence of ET-1, also did not
alter the t-PA response.
|
To determine whether ET-1treated CNS EC also exhibited an increase in
PAI-1 activity, supernatant from 72-hour ET-1treated cultures was
examined for PAI-1 activity. The results in Figure 2
demonstrated that ET-1 increased PAI-1
activity by 63±7%, whereas TNF-
induced a 48±2% increase
compared with untreated cultures (P<0.02.) These data
represent 1 of 3 similar experiments; each experiment was
performed in triplicate.
|
Experiments were performed to determine whether TNF-
augmented the
effects of ET-1 on PAI-1 production. The results in Figure 3
demonstrated that the addition of a
suboptimal concentration of TNF-
(30 pg/mL) to a suboptimal dose of
ET-1 (30 nmol/L) increased PAI-1 production by 92±16%, as
compared with 35±12% with ET-1 alone. This level of PAI-1
production was comparable in magnitude with the maximal effects
observed with optimal concentration of TNF-
(10 ng/mL) (Figure 3
).
|
ET-1 Increases PK-A Activity in CNS EC
We next investigated which signal transduction mechanism was
involved in ET-1induced PAI-1 production. Treatment of CNS EC
with ET-1 for 10 minutes resulted in a concentration-dependent increase
in PK-A activity (Figure 4
). The effect
was significant at 10 nmol/L ET-1, with a maximal 5-fold activation
reached at 100 nmol/L ET-1 (Figure 4
). The time dependence of
PK-A activity in the presence of 100 nmol/L ET-1 is given in Figure 5
. A significant activation of PK-A by
ET-1 was observed after 5 minutes of activation; activity reached a
sharp maximum (5-fold activation) at 10 minutes and declined to the
control levels after 20 minutes of incubation. Intracellular cAMP
concentrations exhibited a similar time and dose response, reaching a
maximum (0.767±0.047 fmol/103 cells; mean±SEM)
at 10 minutes for 100 nmol/L ET-1, with control values of intracellular
cAMP at 0.327±0.031 fmol/103 cells (mean±SEM).
The addition of forskolin and dBu-cAMP was routinely used as a positive
control for both PK-A activity and cAMP elevation. These data
demonstrate that ET-1, as with most reported PK-A
activators, activates PK-A via increased
intracellular cAMP concentrations.
|
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ET-1 Increases PK-C Activity in CNS EC
The time course of the effect of 100 nmol/L ET-1 on membrane PK-C
activity in CNS EC is shown in Figure 6
.
The increase in PK-C activity was significant at 2 minutes, reached a
maximum at 10 minutes, sharply decreased by 20 minutes, and thereafter
slowly declined to the longest observed time of 60 minutes, when the
activity was still above the control levels (Figure 6
). Because
ET-1 activated both PK-A and PK-C in CNS EC, we used the
cAMP-elevating agents forskolin or dBu-cAMP, or the PK-C-activating
agent PMA to test the effects of activation of these signal
transduction pathways on PAI-1 secretion. Figure 7A
shows that both forskolin and dBu-cAMP
dramatically decreased PAI-1 production, whereas activation of
PK-C by PMA resulted in a 2.5-fold increase in PAI-1
production. A combination of both dBu-cAMP and PMA, although
attenuating the effect of PMA alone, resulted in an approximately
2-fold increase in PAI-1 production relative to the control
(Figure 7A
), thus mimicking the effect of ET-1 on the CNS EC.
The role of PK-C activation in ET-1induced PAI-1 production
was established by the use of the specific PK-C inhibitor,
GF. CNS EC exposed to GF for 48 hours demonstrated a marked reduction
in constitutive as well as ET-induced PAI-1 production (Figure 7B
). Cells treated with GF for 48 hours were viable as measured
by the trypan blue dye exclusion assay (>90%), and produced control
levels of the chemokine IL-8 (data not shown), suggesting that the
treated cells were alive and functional. These results demonstrated
that ET-1 stimulation of PAI-1 is mediated through the PK-Cdependent
signaling pathway.
|
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| Discussion |
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induced increase. A lack of
effect of ET-1 (10 nmol/L, 24 hours) on basal t-PA or PAI-1
production by HUVEC was also reported by Rydholm et
al.43 ET-1induced PAI-1 increase was reported not in EC,
but in rat mesangial cells by Iwamoto et al.44
This discrepancy may be because of the duration of the treatment with
ET-1. In agreement with the results of Yamamoto et al42
and Rydholm et al,43 we have also observed no significant
increase of PAI-1 production induced by ET-1 in 24 hours;
however a significant effect was achieved at the 48-hour time, with
further increase relative to the control at 72 hours in the continuous
presence of the peptide. Furthermore, we demonstrated an ET-1induced
increase in PAI-1 activity, which correlated with increased PAI-1
secretion, and no apparent effect on t-PA production.
Our results on the synergistic effect of low ET-1 and TNF-
concentrations to increase PAI-1 production by CNS EC are,
however, in sharp disagreement with the observations of
others42 who reported the attenuation of the
PAI-1increasing effect of TNF-
by ET-1 in HUVEC. One possible
reason for the discrepancy between our results and those above may be
because of the different types of EC used: Yamamoto et
al42 used HUVEC, systemic-derived macrovessels, compared
with CNS-derived microvascular cells, which are functionally
different.45 46 47 For example, coagulation factors, eg,
thrombin, stimulate EC from large vessels but not from the
capillaries48 ; elevation of glucose resulted in decreased
PAI-1 mRNA in brain EC, but not in HUVEC49 ; PMA suppressed
the basic fibroblast growth factor-induced proliferation of
capillary EC, but had no effect on aortic EC50 ; and
vanadate treatment inhibited protein tyrosine kinase activity in aortic
EC, but not capillary EC.51 Thus, these EC differ not only
in the array of growth factor receptors present on their surfaces,
but also in their intracellular regulatory mechanisms. The mechanism of
the synergistic effects of TNF-
and ET-1 are not known, and may be
attributed to several pathways. TNF-
may act indirectly through
products of arachidonic acid metabolism
or directly through intracellular enzymatic mediators (eg, protein
tyrosine kinase). These processes are currently under investigation in
this laboratory.
In the present work we found that ET-1 activates both PK-C- and cAMP-dependent signal transduction pathways. Again, there is no agreement in the literature regarding the effects of ET-1 on intracellular cAMP levels or PK-C activity. The effects of ET-1 on intracellular cAMP levels appear to be cell type-dependent, possibly related to coupling of ETA and ETB receptors to adenylyl cyclases via different G-proteins.52 53 Thus, ET-1 inhibits activator-induced cAMP increase in bovine EC,52 astrocytes,54 rat glioma cells,55 Swiss 3T3 cells,56 pig granulosa cells,57 and in rat nephron segments.58 ET-1induced elevation of cAMP was observed in platelets.59 A small to negligible increase of cAMP by ET-1 was observed in rat glomerular mesangial cells,60 vascular smooth muscle cells,61 and intact preparations of rabbit aorta62 and atria.63 Ladoux and Frelin21 reported that in rat CNS EC, ET-1 inhibits cholera-toxininduced increase in cAMP, whereas Stanimirovic et al64 have shown that in human CNS EC, ET-1 increases intracellular cAMP. Inhibition of cAMP was previously associated with ETB receptors,52 53 and it was indeed thought that EC primarily possess this receptor type.15 16 Our results agree with those of Stanimirovic et al18 and are consistent with the presence of ETA receptors on human CNS EC.
Most studies, including the present work, associate an increase in
intracellular cAMP levels with a decrease in PAI-1 production.
Studying the effect of intracellular cAMP levels on PAI-1
production, Slivka and Loskutoff65 showed that
elevation of cAMP by forskolin and phosphodiesterase
inhibitor 3-isobutyl-1-methyl-xanthene (IBMX) decreases PAI
synthesis by bovine aortic EC. Rydholm et al43 also found
that forskolin decreases PAI-1 production by HUVEC to 61% of
control, as compared with <40% of the control observed by us for the
effect of forskolin or dBu-cAMP (Figure 7A
). Santell and
Levin39 and Francis and Neely66 also reported
that cAMP-elevating agents inhibited the PAI-1 synthesis in HUVEC. In
contrast, only a small effect of forskolin and dBu-cAMP on PAI-1
production by HUVEC was found by Fukao et al.67
Thus, the elevation of cAMP by ET-1 or TNF-
observed in the
present and previous studies cannot explain the increased PAI-1
production.
In addition to activation of PK-A, our data showed that ET-1
activated PK-C in CNS EC, agreeing with the results from other
laboratories using various cell types. ET-1induced release of
inositol 1,4,5-triphosphate, which is an indication of the activity of
phospholipase C, and generation of PK-Cactivating diacylglycerol was
reported in rat CNS EC by Stanimirovic et al.64 In both
smooth muscle cells and fibroblasts, ET-1 rapidly stimulated a biphasic
increase in diacylglycerol that was sustained for 20 minutes or
longer.68 ET-1 stimulated rapid phosphatidylcholine
breakdown in Rat-1 fibroblasts69 and stimulated
phospholipase C in Swiss 3T3 cells56 and in rat vascular
smooth muscle cells leading to PK-C activation.70
ET-1induced PK-C activation was also observed in rat aortic
rings71 and in rat cardiomyocyte
preparations.72 Activation of PK-C by TNF-
was also
demonstrated in several cell lines, including human EC cell line EA.hy
926, HUVEC, and human leukemic cell lines,73 74 75 agreeing
with our observations on human CNS EC. In addition, direct measurements
of PK-C activation of CNS EC at 10 minutes with TNF-
have
demonstrated increased PK-C activity (Zidovetzki et al, unpublished
observation, 1996). However, no effect of TNF-
on PK-C
activity on HUVEC was reported by Ritchie et al.76 Mattila
et al74 explained the lack of PK-C activation by TNF-
reported in some studies by the observation that PK-C activity was
measured at irrelevant times.
In a number of studies, activation of PK-C by PMA or other agents was
associated with increased PAI-1 production in various EC,
including HUVEC,9 43 77 bovine CNS EC,49
bovine aortic EC,49 65 and human CNS EC (this study).
Thus, most of the observations in this and previous studies indicate
that elevated cAMP is associated with a decrease, and activation of
PK-C with an increase, in PAI-1 production. Moreover, Santell
and Levin39 have found that PMA-induced PAI-1 synthesis
was prevented by elevation of cAMP. Nevertheless, Santell and Levin
concluded that activation of cAMP in HUVEC, in coordination with PMA,
led to a profibrinolytic state in EC. A synergistic decrease of PAI-1
production by PMA and forskolin was reported by Sitter et
al.78 Our results in CNS EC subjected to both PK-C
activation by PMA and an increase in cAMP by dBu-cAMP partially agree
with the results of Santell and Levin,39 and support their
conclusion. In our system, dBu-cAMP attenuated PMA-induced PAI-1
production by CNS EC from 254±17% to 212±15%; thus the
combination of both agents still resulted in a significantly higher
PAI-1 production relative to the control (Figure 7A
).
In conclusion, these findings acknowledge the role of ET-1 in regulating hemostasis activity in CNS EC; and further indicate that activation of PK-C is required for ET-1induced increase in PAI-1 production by human CNS EC. With this information, the development of potent ET receptor antagonists or the use of specific signal pathway inhibitors may provide the tools necessary to study ET in cerebral ischemia and provide novel therapeutic approaches.
| Acknowledgments |
|---|
Received July 7, 1998; accepted December 14, 1998.
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