© 1999 American Heart Association, Inc.
Thrombosis |
Endothelin-1 Enhances Plasminogen Activator Inhibitor-1 Production by Human Brain Endothelial Cells via Protein Kinase C-Dependent Pathway
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 |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—The effects of endothelin-1 (ET-1) on the production of plasminogen activator inhibitor 1 (PAI-1) and tissue plasminogen activator (t-PA) by human brain-derived endothelial cells in culture were studied. At 100 nmol/L, ET-1 increased PAI-1 production by 88±6% within 72 hours, and increased PAI-1 mRNA expression within 1 hour of stimulation; there was no significant effect on t-PA production. PAI-1 activity was also examined and found to increase with ET-1 treatment. Suboptimal concentrations of ET-1 and tumor necrosis factor-
(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 |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Increased levels of endothelin-1 (ET-1) in plasma or cerebrospinal fluid of patients with hypertension, ischemic stroke, and subarachnoid hemorrhage have implicated ET-1 as a mediator of cerebrovascular responses in these disorders.1 2 3 4 The endothelins (ETs) are a family of potent vasoactive peptides of 21 amino acids in length5 ; cDNA cloning revealed 3 isopeptides, ET-1 to ET-3.6 ET has been shown to induce vasoconstriction,5 but has numerous other biological actions, including bronchoconstriction and hormone secretion,7 8 9 and acts on a variety of cell types, including endothelial cells (EC) and astrocytes.10 11 12 Two distinct ET receptor subtypes, ETA and ETB, have been cloned and sequenced.13 14 These receptors contain 7 hydrophobic transmembrane domains common to the superfamily of G-protein-coupled receptors; ETA shows selective activity for ET-1 and ET-213 whereas ETB shows nonselective affinity for all 3 isopeptides.14 EC possess predominantly ETB receptors, which mediate vasodilation.15 16 It has been shown that the ETA receptor is expressed on vascular smooth muscle cells and human brain EC, and mediates vasoconstriction.17 18 Both receptor subtypes are functionally coupled to phospholipase C via GTP-binding proteins in EC.19 Signal transduction on ET-1 binding to its receptor may differ among species and tissues and includes activation of phospholipase C and protein kinase C, mobilization of Ca2+ from intracellular stores, and influx of extracellular Ca2+, as well as inhibition of agonist-stimulated cAMP production.20 21 22
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 |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Reagents
Protein kinase A (PK-A) and PK-C assay kits were purchased from Gibco Life Technologies; cAMP assay kit from DuPont company; [32P]ATP from ICN. Bisindolylmaleimide-GF-109203X (GF; PK-C inhibitor) was purchased from Calbiochem. Phorbol-12-myristate-13-acetate (PMA), dibutyryl cAMP (dBu-cAMP), and forskolin were obtained from Sigma. TNF-
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-A–specific 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 |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
ET-1 Upregulates PAI-1 Production by CNS EC
To test the hypothesis that ET-1 directly affects hemostasis, CNS EC were exposed to ET-1 at concentrations of 30 and 100 nmol/L for 72 hours and tested for PAI-1 production with ELISA. The results show that ET-1 significantly (P<0.02) increased PAI-1 production in a dose-dependent manner (Figure 1A
). To determine whether the observed
increase represented an upregulation in PAI-1 mRNA
expression, RPA was performed. The results (Figure 1B)
demonstrated that ET-1 upregulated PAI-1 mRNA by 80% after 1 hour of
ET-1 treatment; this level was maintained at 3 and 6 hours, and then
returned to baseline 24 hours after stimulation. To determine whether
t-PA production was also altered by ET-1, culture supernatants
were treated as described above and examined for t-PA with ELISA
(Figure 1C). The results showed that t-PA was not affected by
ET-1 at the optimal concentration used (100 nmol/L) (Figure 1C).
The proinflammatory cytokine TNF-, 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-1–treated CNS EC also exhibited an increase in
PAI-1 activity, supernatant from 72-hour ET-1–treated 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-1–induced 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.
|
|
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-1–induced 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-C–dependent
signaling pathway.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
This work was initiated to examine the effect of ET-1 on the coagulation activity of human CNS EC, and to identify the intracellular signal transduction pathways responsible for this effect. The regulation of PAI-1 secretion by ET-1 appears to be a complex process, which will be best discussed by separating it into different parts, because there is no agreement in the literature on such important questions as the effect of ET-1 on PAI-1 or t-PA production, the effect of ET-1 on intracellular cAMP level, and the effect of increasing cAMP or PK-C activation on the production of PAI-1. First, there is no agreement in the literature on the effect of ET-1 on PAI-1 production. Previous studies of the effects of ET-1 on t-PA and PAI-1 production by EC failed to detect ET-1–induced increase in PAI-1 production. Yamamoto et al42 found that ET-1 did not affect PAI-1 released from human umbilical vein endothelial cells (HUVEC), but suppressed thrombin-induced release of PAI-1 from HUVEC, and only at high concentrations of both thrombin and ET-1. Yamamoto et al42 thus suggested that ET-1 may serve as an anti-fibrinolytic factor when EC-mediated fibrinolysis is stimulated by thrombin; they also suggested that both vasoconstriction and reduction of fibrinolysis are physiological roles of ET-1. This group also reported that although 100 nmol/L ET-1 did not affect PAI-1 secretion from HUVEC in 24 hours of incubation, it significantly attenuated the TNF-
–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-1–induced 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-1–induced
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-1–increasing 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-1–induced 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-toxin–induced 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-1–induced release of
inositol 1,4,5-triphosphate, which is an indication of the activity of
phospholipase C, and generation of PK-C–activating 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-1–induced 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-1–induced 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 |
|---|
The study was supported by the National Institutes of Health grant P01-NS 31946.
Received July 7, 1998; accepted December 14, 1998.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Kohno M, Yasunari K, Murakawa K, Yokokawa K, Horio T, Fukui T, Takeda T. Plasma immunoreactive endothelin in essential hypertension. Am J Med. 1990;88:614–618.[Medline] [Order article via Infotrieve]
2.
Lampl Y, Fleminger G, Gilad R, Galron R, Sarova-Pinhas
I, Sokolovsky, M. Endothelin in cerebrospinal fluid and plasma of
patients in the early stage of ischemic stroke.
Stroke. 1997;28:1951–1954.
3. Masaoka H, Suzuki R, Hirata Y, Emori T, Marumo F, Hirakawa K. Raised plasma endothelin in aneurysmal subarachnoid haemorrhage. Lancet. 1989;2:1402.[Medline] [Order article via Infotrieve]
4. Suzuki H, Sato S, Suzuki Y, Takekoshi K, Ishihara N, Shimoda S. Increased endothelin concentration in CSF from patients with subarachnoid hemorrhage. Acta Neurol Scand. 1990;81:553–554.[Medline] [Order article via Infotrieve]
5. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. 1988;332:411–415.[Medline] [Order article via Infotrieve]
6.
Inoue A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T,
Goto K, Masaki T. The human endothelin family: three structurally and
pharmacologically distinct isopeptides predicted by three separate
genes. Proc Natl Acad Sci U S A. 1989;86:2863–2867.
7.
de Nucci G, Thomas R, D'Orleans-Juste P, Antunes
E, Walder C, Warner TD, Vane JR. Pressor effects of circulating
endothelin are limited by its removal in the pulmonary
circulation and by the release of prostacyclin and
endothelium-derived relaxing factor. Proc Natl
Acad Sci U S A. 1988;85:9797–9800.
8. Galron R, Kloog Y, Bdolah A, Sokolovsky M. Functional endothelin/sarafotoxin receptors in rat heart myocytes: structure-activity relationships and receptor subtypes. Biochem Biophys Res Commun. 1989;163:936–943.[Medline] [Order article via Infotrieve]
9. Fukuda Y, Hirata Y, Yoshimi H, Kojima T, Kobayashi Y, Yanagisawa M, Masaki T. Endothelin is a potent secretagogue for atrial natriuretic peptide in cultured rat atrial myocytes. Biochem Biophys Res Commun.1988;155:167–172.
10. Sakurai T, Yanagisawa M, Takuwa Y, Miyazaki H, Kimura S, Goto K, Masaki T. Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature. 1990;348:732–735.[Medline] [Order article via Infotrieve]
11. Masaki T, Vane JR, Vanhoutte PM. International Union of Pharmacology nomenclature of endothelin receptors. Pharmacol Rev. 1994;46:137–142.[Medline] [Order article via Infotrieve]
12.
Mac Cumber MW, Ross CA, Snyder SH. Endothelin in brain:
receptor, mitogenesis, and biosynthesis in glial cells. Proc Natl
Acad Sci U S A. 1990;87:2359–2363.
13. Arai H, Hori S, Aramori I, Ohkubo H, Nakanishi S. Cloning and expression of a cDNA encoding an endothelin receptor. Nature. 1990;348:730–732.[Medline] [Order article via Infotrieve]
14. Sakurai T, Yanagisawa M, Masaki T. Molecular characterization of endothelin receptors. Trends Pharmacol Sci. 1992;13:103–108.[Medline] [Order article via Infotrieve]
15. Takayanagi R, Kitazumi K, Takasaki C, Ohnaka K, Aimoto S, Tasaka K, Ohashi M, Nawata H. Presence of non-selective type of endothelin receptor on vascular endothelium and its linkage to vasodilation. FEBS Lett. 1991;282:103–106.
16. Emori T, Hirata Y, Kanno K, Ohata K, Eguchi S, Imai T, Schichiri M, Marumo F. Endothelin-3 stimulates production of endothelium-derived nitric oxide via phosphoinositide breakdown. Biochem Biophys Res Commun. 1991;174:228–235.[Medline] [Order article via Infotrieve]
17. Miller RC, Pelton JT, Huggins JP. Endothelins—from receptors to medicine. Trends Pharmacol Sci. 1993;14:54–60.[Medline] [Order article via Infotrieve]
18. Stanimirovic DB, Yamamoto T, Uematsu S, Spatz M. Endothelin-1 receptor binding and cellular signal transduction in cultured human brain endothelial cells. J Neurochem. 1994;62:592–601.[Medline] [Order article via Infotrieve]
19. Eguchi S, Kozuka M, Hirose S, Ito T, Hirata Y, Marumo F, Hagiwara H. Identification of G-protein-coupled endothelin receptors in cultured bovine endothelial cells. Biochem Biophys Res Commun. 1991;174:1343–1346.[Medline] [Order article via Infotrieve]
20. Xuan YT, Whorton AR, Watkins WD. Inhibition by nicardipine of endothelin-mediated inositol phosphate formation and Ca2+ mobilization in smooth muscle cell. Biochem Biophys Res Commun. 1989;160:758–764.[Medline] [Order article via Infotrieve]
21. Ladoux A, Frelin C. Endothelins inhibit adenylate cyclase in brain capillary endothelial cells. Biochem Biophys Res Commun. 1991;180:169–173.[Medline] [Order article via Infotrieve]
22.
Gardner JP, Tokudome G, Tomonari H, Maher E, Hollander
D, Aviv A. Endothelin-induced calcium responses in human vascular
smooth muscle cells. Am J Physiol. 1992;262:C148–C155.
23. Plow EF, Merren T, Redlitz A, Mills LA, Hoover-Plow JL. The cell biology of the plasminogen system. FASEB J. 1995;9:939–945.[Abstract]
24. Hekman CM, Loskutoff DJ. Kinetic analysis of the interactions between plasminogen activator inhibitor 1 and both urokinase and tissue plasminogen activator. Arch Biochem Biophys. 1988;262:199–210.[Medline] [Order article via Infotrieve]
25. Loskutoff DJ, Sawdey M, Mimuro J. Type 1 plasminogen activator inhibitor. Prog Hemostasis Thromb. 1989;9:87–115.[Medline] [Order article via Infotrieve]
26.
Sprengers ED, Kluft C. Plasminogen
activator inhibitors. Blood. 1987;69:381–387.
27. Loskutoff DJ, Sawdey M, Keeton M, Schneiderman J. Regulation of PAI-1 gene expression in vivo. Thromb Haemostasis. 1993;70:135–137.[Medline] [Order article via Infotrieve]
28. Hamsten A, Wiman B, de Faire, U, Blomback M. Increased plasma levels of a rapid inhibitor of myocardial infarction. N Engl J Med. 1985;313:1557–1563.[Abstract]
29. Gram J, Jespersen J, Kluft C, Rijken DC. On the usefulness of fibrinolysis variables in the characterization of a risk group for myocardial reinfarction. Acta Med Scand. 1987;221:149–153.[Medline] [Order article via Infotrieve]
30. Wiman B, Ljungberg B, Chmielewska J, Urden G, Blomback M, Johnsson H. The role of the fibrinolytic system in deep vein thrombosis. J Lab Clin Med. 1985;105:265–270.[Medline] [Order article via Infotrieve]
31.
Schneiderman J, Sawdey MS, Keeton MR, Bordin GM,
Bernstein EF, Dilley RB, Loskutoff DJ. Increased type 1
plasminogen activator inhibitor
gene expression in atherosclerotic human arteries. Proc Natl Acad
Sci U S A. 1992;89:6998–7002.
32.
Lupu F, Bergonzelli GE, Heim DA, Cousin E, Genton CY,
Bachmann F, Kruithof EKO. Localization and production of
plasminogen activator inhibitor-1
in human healthy and atherosclerotic arteries. Arterioscler
Thromb. 1993;13:1090–1100.
33. Schleef RR, Loskutoff DJL. Fibrinolytic system of vascular endothelial cells. Haemostasis. 1988;18:328–341.[Medline] [Order article via Infotrieve]
34.
Van Mourik JA, Lawrence DA, Loskutoff DJ. Purification
of an inhibitor of plasminogen
activator (antiactivator) synthesized by
endothelial cells. J Biol Chem. 1984;259:14914–14921.
35. Levin EG, Marzec U, Anderson J, Harker LA. Thrombin stimulates tissue plasminogen activator release from cultured human endothelial cells. J Clin Invest. 1984;74:1988–1995.
36. Heaton JH, Dame MK, Gelehrter TD. Thrombin induction of plasminogen activator, inhibitor mRNA in human umbilical vein endothelial cells in culture. J Lab Clin Med. 1992;120:222–228.[Medline] [Order article via Infotrieve]
37. Hanss M, Collen D. Secretion of tissue-type plasminogen activator and plasminogen activator inhibitor by cultured human endothelial cells: modulation by thrombin, endotoxin, and histamine. J Lab Clin Med. 1987;109:97–104.[Medline] [Order article via Infotrieve]
38.
Crutchley DJ, Conanan LB. Endotoxin induction of an
inhibitor of plasminogen activator
in bovine pulmonary artery endothelial cells.
J Biol Chem. 1986;261:154–159.
39.
Santell L, Levin EG. Cyclic AMP potentiates phorbol
ester stimulation of tissue, plasminogen
activator release and inhibits secretion of
plasminogen activator inhibitor-1
from human endothelial cells. J Biol
Chem.. 1988;263:16802–16808.
40. Vassalli P. The pathophysiology of tumor necrosis factors. Annu Rev Immunol. 1992;10:411–452.[Medline] [Order article via Infotrieve]
41. Hofman FM, Dohadwala MH, Wright AD, Hinton DR, Walker SM. Exogenous tat protein activates central nervous system-derived endothelial cells. J Neuroimmunol. 1994;54:19–24.[Medline] [Order article via Infotrieve]
42. Yamamoto C, Kaji T, Sakamoto M, Kozuka H. Modulation by endothelin-1 of tissue plasminogen activator and plasminogen activator inhibitor-1 release from cultured human vascular endothelial cells: interaction of endothelin-1 with cytokines. Biol Pharm Bull. 1993;16:714–715.[Medline] [Order article via Infotrieve]
43. Rydholm H, Boström S, Eriksson E, Risberg B. Complex intracellular signal transduction regulates tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1) synthesis in cultured human umbilical vein endothelium. Scand J Clin Lab Invest. 1990;55:323–330.
44. Iwamoto T, Tamaki K, Nakayama M, Okuda S, Fujishima M. Effect of endothelin 1 on fibrinolysis and plasminogen activator inhibitor 1 synthesis in rat mesangial cells. Nephron. 1996;73:273–279.[Medline] [Order article via Infotrieve]
45. Bar RS, Boes M, Dake BL, Booth BA, Henley SA, Sandra A. Insulin, insulin-like, growth factors, and vascular endothelium. Am J Med.. 1988;85:59–70.[Medline] [Order article via Infotrieve]
46. Boes M, Dake BL, Bar RS. Interactions of cultured endothelial cells with TGF-ß, bFGF, PDGF, and IGF-1. Life Sci. 1991;48:811–821.[Medline] [Order article via Infotrieve]
47.
Beitz JG, Kim I-S, Calabresi P, Fraskelon AR. Human
microvascular endothelial cells express receptors for
platelet-derived growth factor. Proc Natl Acad Sci
U S A. 1991;88:2021–2025.
48. Dupuy E, Bikfalvi A, Rendu F, Toledano SL, Tobelem G. Thrombin mitogenic responses and protein phosphorylation are different in cultured human endothelial cells derived from large and microvessels. Exp Cell Res. 1991;185:363–372.
49. Kollros PR, Konkle BA, Ambarian AP, Henrikson P. Plasminogen activator inhibitor-1 expression by brain microvessel endothelial cells is inhibited by elevated glucose. J Neurochem. 1994;63:903–909.[Medline] [Order article via Infotrieve]
50.
Doctrow SR, Folkman J. Protein kinase C
activators suppress stimulation of capillary
endothelial cell growth by angiogenic
endothelial mitogens. J Cell Biol.. 1987;104:679–687.
51. Maher PA. Stimulation of endothelial cell proliferation by vanadate is specific for microvascular endothelial cells. J Cell Physiol. 1992;151:549–554.[Medline] [Order article via Infotrieve]
52.
Eguchi S, Hirata Y, Imai T, Marumo F. Endothelin
receptor subtypes are coupled, to adenylate cyclase via
different guanyl nucleotide-binding proteins in
vasculature. Endocrinology. 1993;132:524–529.
53.
Aramori I, Nakanishi S. Coupling of two endothelin
receptor subtypes to differing signal transduction in transfected
Chinese hamster ovary cells. J Biol Chem. 1992;267:12468–12474.
54. Martin P, Delumeau JC, Durieu-Trautmann O, Le Nguyen D, Premont J, Strosberg AD, Couraud PO. Are several G proteins involved in the different effects of endothelin-1 in mouse striatal astrocytes? J Neurochem. 1991;56:1270–1275.[Medline] [Order article via Infotrieve]
55. Couraud PO, Durieu-Trautmann O, Le Nguyen D, Marin P, Glibert F, Strosberg AD. Functional endothelin-1 receptors in rat astrocytoma C6. Eur J Pharmacol. 1991;206:191–198.[Medline] [Order article via Infotrieve]
56.
Takuwa N, Takuwa Y, Yanagisawa M, Yamashita K, Masaki
T. A novel, vasoactive peptide endothelin stimulates mitogenesis
through inositol lipid turnover in Swiss 3T3 fibroblasts. J
Biol Chem. 1989;264:7856–7861.
57.
Iwai M, Hasegawa M, Taii S, Sagawa N, Nakao K, Imura H,
Nakanishi S, Mori T. Endothelins inhibit luteinization of cultured
porcine granulosa cells. Endocrinology. 1991;129:1909–1914.
58. Tomita K, Nonoguchi H, Marumo F. Effects of endothelin on peptide-dependent cyclic adenosine monophosphate accumulation along the nephron segments of the rat. J Clin Invest. 1990;185:2014–2018.
59. Thiemermann C, Lidbury PS, Thomas GR, Vane JR. Endothelin inhibits ex vivo platelet aggregation in the rabbit. Eur J Pharmacol. 1988;158:181–185.[Medline] [Order article via Infotrieve]
60. Simonson MS, Dunn MJ. Endothelin-1 stimulates contraction of rat glomerular mesangial cells and potentiates beta-adrenergic-mediated cyclic adenosine monophosphate accumulation. J Clin Invest. 1990;85:790–797.
61. Sugiura M, Inagami T, Harem GMT, Johns JA. Endothelin action: inhibition by a protein kinase C inhibitor and involvement of phosphoinositols. Biochem Biophys Res Commun. 1989;158:170–176.[Medline] [Order article via Infotrieve]
62.
Ohlstein EH, Horohonich S, Hay DWP. Cellular mechanisms
of endothelin in rabbit aorta. J Pharmacol Exp Ther. 1989;250:548–555.
63. Vigne P, Lazdunski M, Frelin C. The inotropic effect of endothelin-1 on rat atria involves hydrolysis of phosphatidylinositol. FEBS Lett. 1989;249:143–146.[Medline] [Order article via Infotrieve]
64. Stanimirovic DB, Nikodijevic B, Nikodijevic-Kedeva D, McCarron RM, Spatz M. Signal transduction and Ca2+ uptake activated by endothelins in rat brain endothelial cells. Eur J Pharmacol. 1994;288:1–8.[Medline] [Order article via Infotrieve]
65. Slivka SR, Loskutoff DJ. Regulation of type I plasmonogen activator inhibitor synthesis by protein kinase C and cAMP in bovine aortic endothelial cells. Biochim Biophys Acta. 1991;1094:317–322.[Medline] [Order article via Infotrieve]
66. Francis RB, Neely S. Inhibition of endothelial secretion of tissue-type plasminogen activator and its rapid inhibitor by agents which increase intracellular cyclic AMP. Biochim Biophys Acta. 1989;1012:207–213.[Medline] [Order article via Infotrieve]
67. Fukao H, Matsumoto H, Ueshima S, Okada K, Matsuo O. Effects of fibrin on the secretion of plasminogen activator inhibitor-1 from endothelial cells and on protein kinase C. Life Sci. 1995;57:1267–1276.[Medline] [Order article via Infotrieve]
68.
Muldoon L, Rodland KD, Forsythe ML, Magun BE.
Stimulation of phosphatidylinositol hydrolysis, diacylglycerol release,
and gene expression in response to endothelin, a potent new agonist for
fibroblasts and smooth muscle cells. J Biol Chem. 1989;264:8529–8536.
69. van der Bend RL, de Widt J, van Corven EJ, Moolenaar WH, van Blitterswijk WJ. The biologically active phospholipid, lysophosphatidic acid, induces phosphatidylcholine breakdown in fibroblasts via activation of phospholipase D: comparison with the response to endothelin. Biochem J. 1992;285:235–240.
70.
Xuan YT, Wang OL, Whorton AR. Regulation of
endothelin-induced Ca2+ mobilization in smooth
muscle cells by protein kinase C. Am J Physiol. 1994;266:C1560–C1567.
71.
Wright HM, Malik KU. Prostacyclin synthesis elicited by
endothelin-1 in rat aorta is mediated by an ETA
receptor via influx of calcium and is independent of protein kinase C.
Hypertension. 1995;26:1035–1040.
72.
Shubeita HE, Martinson EA, Van Bilsen M, Chien KR,
Brown JH. Proc Natl Acad Sci U S A. 1992;89:1305–1309.
73. Brenner DA, O'Hara M, Angel P, Chojkier M, Karin M. Prolonged activation of jun and collagenase genes by tumour necrosis factor-alpha. Nature. 1989;337:661–663.[Medline] [Order article via Infotrieve]
74. Mattila P, Majuri ML, Mattila PS, Renkonen R. TNF alpha-induced expression of endothelial adhesion molecules, ICAM-1 and VCAM-1, is linked to protein kinase C activation. Scand J Immunol. 1992;36:159–165.[Medline] [Order article via Infotrieve]
75.
Meichle A, Schutze S, Hensel G, Brunsing D, Kronke M.
Protein kinase, C-independent activation of nuclear factor kappa B by
tumor necrosis factor. J Biol Chem. 1990;265:8339–8343.
76. Ritchie AJ, Johnson DR, Ewenstein BM, Pober JS. Tumor necrosis factor induction of endothelial cell surface antigens is independent of protein kinase C activation or inactivation: studies with phorbol myristate acetate and staurosporine. J Immunol. 1991;146:3056–3062.[Abstract]
77.
Konkle BA, Kollros PR, Kelly MD. Heparin-binding growth
factor-1 modulation of plasminogen activator
inhibitor-1 expression. Interaction with cAMP and protein
kinase C-mediated pathways. J Biol Chem. 1990;265:21867–21873.
78.
Sitter, T, Toet K, Fricke, H, Schiffl H, Held E,
Kooistra T. Modulation of procoagulant and fibrinolytic system
components of mesothelial cells by inflammatory mediators.
Am J Physiol. 1996;271:R1256–R1263.
This article has been cited by other articles:
 |
|
 |
|
  J. Milan, C. Charalambous, R. Elhag, T. C. Chen, W. Li, S. Guan, F. M. Hofman, and R. Zidovetzki Multiple signaling pathways are involved in endothelin-1-induced brain endothelial cell migration Am J Physiol Cell Physiol, July 1, 2006; 291(1): C155 - C164. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Eddy Plasminogen activator inhibitor-1 and the kidney Am J Physiol Renal Physiol, August 1, 2002; 283(2): F209 - F220. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Uchiyama, M. Kurabayashi, Y. Ohyama, T. Utsugi, N. Akuzawa, M. Sato, S. Tomono, S. Kawazu, and R. Nagai Hypoxia Induces Transcription of the Plasminogen Activator Inhibitor-1 Gene Through Genistein-Sensitive Tyrosine Kinase Pathways in Vascular Endothelial Cells Arterioscler Thromb Vasc Biol, April 1, 2000; 20(4): 1155 - 1161. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||