(Received for publication, July 14, 1995; and in revised form, March 13, 1996)
From the
The presence of prostaglandin (PG) H in the
supernatant of human umbilical vein endothelial cells (HUVEC)
stimulated by thrombin restores the capacity of aspirin-treated
platelets to generate thromboxane (TX) B
. Induction of
cyclooxygenase-2 (Cox-2) by interleukin (IL)-1
or a phorbol ester
increases this formation. HUVEC treated with aspirin lost their
capacity to generate PGs but recovery occurred after 3- or 6-h
induction of Cox-2 with phorbol ester or IL-1
. Enzyme activity of
the newly synthesized Cox-2 in aspirin-treated cells, evaluated after
immunoprecipitation, was similar to untreated cells but after 18 h of
cell stimulation only 50-60% recovery of Cox-1 was observed. The
use of SC58125, a selective Cox-2 inhibitor, confirmed these findings
in intact cells. Cyclooxygenase activity was related to the amount of
Cox proteins present in the cells, but after induction of Cox-2,
contribution of the latter to PG production was 6-8-fold that of
Cox-1. Aspirin-treated or untreated cells were incubated in the absence
or presence of SC58125 and stimulated by thrombin, the ionophore
A23187, or exogenous arachidonic acid. The production of endogenous
(6-keto-PGF
, PGE
, PGF
) versus transcellular (TXB
) metabolites was
independent of the inducer, the source of arachidonic acid and the Cox
isozyme. However, in acetylsalicylic acid-treated cells, after 6-h
stimulation with IL-1
, newly synthesized Cox-2 produced less
TXB
than 6-keto-PGF
compared to untreated
cells. At later times (>18 h), there was no metabolic difference
between the cells. These studies suggest that in HUVEC, Cox
compartmentalization occurring after short-term activation may
selectively affect transcellular metabolism, but not constitutive
production, of PGs.
Thromboxane (TX) ()A
and prostaglandin
(PG) I
(prostacyclin) are the main products of arachidonic
acid metabolism via the cyclooxygenase (Cox; also known as PGH synthase
(EC 1.14.99.1)) pathway in platelets and endothelial cells,
respectively(1) . The synthesis of TXA
is
suppressed by aspirin (ASA), an irreversible Cox
inhibitor(2, 3) . Some years ago, Marcus and
colleagues (4) demonstrated that endoperoxides generated by
platelets stimulated by thrombin, collagen, or the Ca
ionophore A23187 could restore the capacity of ASA-treated
endothelial cells to synthesize PGI
, as hypothesized
earlier(5) . Because platelets are non-nucleated cells, their
potential for producing TXA
is irreversibly suppressed in vitro or in vivo during their lifetime. Whereas
platelets contain a constitutively expressed Cox (Cox-1), in most other
cells the existence of an inducible Cox has been demonstrated. This
novel PGH synthase 2, i.e. Cox-2, is highly regulated by
external ligands such as cytokines, tumor promoters, or growth
factors(6) . Cox-2 was identified as a separate product of an
early response gene(7, 8) . It has been cloned and
sequenced in human endothelial cells(9, 10) . In
addition to rapid renewal of the enzyme, induction of Cox-2 has been
regarded as a mechanism by which cells increase their capacity to
synthesize PGs in excess of that provided by Cox-1(11) .
However, metabolic (12) and immunofluorescence localization
experiments (13) suggest that substrate and/or enzyme
compartmentalization may constitute an additional important step in
regulation of PG production.
Here we show that endothelial cells
stimulated by thrombin can restore the production of TX by
aspirin-treated platelets via transcellular metabolism. Both Cox
isozymes contribute to the capacity of endothelial cells to synthesize
PGH, from endogenous or exogenous arachidonic acid. The net
amount of products synthesized was dependent on the quantity of active
cyclooxygenases in cells, and Cox compartmentalization occurring after
short-term activation transiently affects transcellular metabolism, but
not constitutive production, of PGs.
In some experiments, endothelial
cells were incubated for 30 min with 100 µM ASA, SA, or
vehicle (i.e. ethanol). After removal of the supernatant,
cells were washed 3 times and maintained for 30 min in culture medium
in order to remove any residual ASA or SA. The medium was changed and
cells were activated with PMA (20 nM), IL-1 (25
units/ml), or medium alone (containing 5% NHS), for different times.
Cells were either lysed for Western blot analysis (see below) or
incubated with aspirin-treated platelets and stimulated by thrombin,
arachidonic acid, or the Ca
ionophore A23187 in the
absence or presence of SC58125, a specific Cox-2
inhibitor(18) . The supernatant was assayed for TXB
and 6-keto-PGF
.
In preliminary experiments, the specificity of the TXB determination by EIA was verified by purifying the samples by
thin-layer chromatography or HPLC. In these experiments,
[
H]TXB
was added before solid-phase
extraction to estimate the losses occurring during the various steps of
purification. HPLC was done as described above. The fractions
corresponding to TXB
were pooled, evaporated to dryness,
and EIA buffer was added. Thin-layer chromatography was performed after
developing the plates in chloroform/methanol/acetic acid/water
(90:8:1:0.8, v/v). Authentic TXB
(1-2 µg) was
applied in a separate lane and visualized using a 3.5% phosphomolybdic
acid spray. The corresponding lane was scraped and compounds were
eluted by adding the silica to EIA buffer (0.1 M phosphate, pH
7.4, containing 0.15 M NaCl, 0.1% bovine serum albumin, and
0.01% sodium azide). Results after either purification technique
corresponded to those obtained by direct EIA.
Figure 1:
Synthesis of TX in
co-incubations of endothelial cells and aspirin-treated platelets after
2-min stimulation by thrombin. A, various combinations of
cells. Endothelial cells (EC) (2 10
cells
in 12-well dishes) or platelets (PL) alone, or in combination
(PL/EC = 125), were stimulated with thrombin (2 units/ml) and
TXB
was measured by EIA. In some incubates, EC were
activated for 6 h with 20 nM PMA prior to addition of
platelets. In all incubations, platelets were pretreated with ASA (see
``Experimental Procedures''), but in the data presented in
the last histogram ASA (100 µM) was added to endothelial
cells prior to co-incubation. B, effect of platelet
concentration. Endothelial cells (as above) activated by PMA were
incubated with increasing numbers of platelets and stimulated with
thrombin. C, concentration-response effect of thrombin.
Co-incubations (same as above) were stimulated with increasing
concentrations of thrombin. The supernatants were acidified with citric
acid (final concentration, 50 mM), neutralized, and assayed by
EIA. Data represent the mean ± S.D. (three to four experiments
in replicates of 2).
The dependence of
platelets was tested as a function of increases in the ratio of
platelets to endothelial cells. The formation of TXB increased as a function of platelet number and a plateau was
reached at 0.5-1
10
platelets/well (i.e. endothelial cell/platelet ratio of 1/250, Fig. 1B). No TX was formed in the absence of thrombin
stimulation; TXB
, although, increased as a function of
thrombin concentration (0-4 units/ml) (Fig. 1C).
In subsequent experiments, respective platelet/endothelial cell ratios
of 125 and 2 units/ml of thrombin were used.
Figure 2:
Synthesis of TX by aspirin-treated
platelets from PGH released from thrombin-stimulated
endothelial cells. In all experiments, endothelial cells were activated
for 6 h with 20 nM PMA and thrombin was used at 2 units/ml. A
ratio of PL/EC = 125 was used in co-incubations. A and B, presence of a metabolic intermediate in the supernatant of
thrombin-stimulated endothelial cells. Activated endothelial cells in
100-mm
culture dishes (2.5
10
cells)
were stimulated for 30 s by thrombin. A, the supernatant was
transferred to 150 µl of aspirin-treated platelet suspension (final
concentration, 0.2
10
cells/ml) or to buffer for 2
min. The reaction was quenched and the incubate analyzed as described
in the legend to Fig. 1(mean ± S.D. of three separate
experiments). B, the supernatant was transferred to a
polypropylene tube and at the indicated times an aliquot (150 µl)
was added to 150 µl of platelets (final, 0.2
10
cells/ml) and incubated for 2 min. The apparent half-life of the
metabolic intermediate was calculated from the formation of TX in each
experiment (n = 5). C, IgGs directed against a
PGH
analog decreased the synthesis of TX from a
co-incubation of platelets with endothelial cells. Co-incubation and
stimulation by thrombin (2 min) were carried out in the presence of
increasing amounts of IgGs directed against a stable PGH
mimic (
). Analysis of 6-keto-PGF
(
) was performed in the same samples. Nonspecific IgGs
were also used as controls and the effect of the highest concentration
(150 µg/ml) is shown (
). The curve corresponds to the mean of
two separate experiments. D, effect of a TX synthase inhibitor
on TXB
produced during co-incubation of platelets with
HUVEC. Co-incubation of cells and stimulation were performed as
previously, in the presence of increasing concentrations of
furegrelate.
, TXB
;
, PGE
. The
results are the mean ± S.D. of three different
experiments.
Figure 3:
HPLC analysis of the supernatant of
[H]arachidonic acid-labeled endothelial cells in
the absence (A) or presence (B) of aspirin-treated
platelets. PMA-activated HUVEC (6
10
cells) grown
in T-162 flasks were labeled with [
H]arachidonic
acid as described under ``Experimental Procedures.'' Cells
were stimulated for 2 min by 2 units/ml thrombin in the absence or
presence of platelets and analysis of samples by HPLC was performed as
described under ``Experimental Procedures.'' Each fraction
(0.33 min) was counted for radioactivity, and immunoreactivity was
analyzed by EIA.
Figure 4:
Relation between cyclooxygenase activity
of endothelial cells (i.e. 6-keto-PGF) and TX
synthesis in a co-incubation of ASA-treated platelets with endothelial
cells. HUVEC (1.9
10
cells) were activated by PMA
(0-20 nM) or IL-1
(0-25 units/ml) for various
times (1.5-24 h) in order to obtain various levels of Cox-2.
After addition of ASA-treated platelets (platelet/EC = 125),
cell co-incubates were stimulated as described in previous figure
legends. TXB
and 6-keto-PGF
were
determined in the same samples.
Figure 5:
Western blot analysis of Cox-2 expression
after treatment of endothelial cells with vehicle, ASA, or SA followed
by IL-1 activation. HUVEC were incubated for 30 min in the absence
or presence of ASA or SA (100 µM each). The efficiency of
ASA treatment was verified by measuring the incapacity of the cells to
produce 6-keto-PGF
immediately after the 30-min
incubation. Cells were washed as described under ``Experimental
Procedures'' and activated with 25 units/ml IL-1
for 6 h or
incubated in NHS alone. Lanes show control cells incubated in
the absence or presence of IL-1
, respectively, or after
pretreatment with SA or ASA. After incubation, cell monolayers
(3-4
10
cells/well) were lysed and Cox-2 was
analyzed after SDS-PAGE as described under ``Experimental
Procedures.'' This figure is representative of four experiments.
-Actin was used in all samples as an internal standard for protein
load correction.
Figure 6:
Cox activities in the immunoprecipitates
of Cox-2 obtained after 3 (PMA) or 6 (IL-1) h activation of
endothelial cells, following treatment of cells with ASA or SA.
Incubations were carried out as described in the legend to Fig. 5. After activation, the cells were lysed and Cox-2 was
immunoprecipitated as described under ``Experimental
Procedures.'' Cyclooxygenase activity was evaluated by measuring
PGE
in the supernatant of the immunoprecipitates after
addition of arachidonic acid (10 µM). The histograms on
the left show the activity in immunoprecipitates of cells
immediately after treatment with SA or ASA. Results represent the mean
± S.E. of three different
experiments.
However, in intact cells less
TX was formed after activation of aspirin-treated endothelial cells (Fig. 7A). The specific Cox-2 inhibitor SC58125 was
used in order to identify the enzyme responsible for the recovery in
activity after treatment with ASA. This compound totally suppressed the
synthesis of TX in cells treated with ASA, prior to activation. There
was no difference in TXB formation in SA-treated and
untreated cells (Fig. 7B). These results demonstrate
that: 1) after ASA treatment, the rapid recovery of enzyme activity
responsible for TX synthesis is due exclusively to Cox-2; 2) in
untreated cells (or cells treated with SA), Cox-1 and Cox-2 are
involved in transcellular biosynthesis; 3) in control cells (incubated
in NHS), a significant amount of TX originates from Cox-2 (50-70%
of total) (Fig. 7B, left panel). This last result
indicates that induction of Cox-2 increases 6-8-fold the basal
capacity (i.e. provided by Cox-1) of endothelial cells to
generate PGs. Aspirin-treated cells stimulated for 18 h with IL-1
recovered 92 ± 20% of TX synthesis compared to untreated cells (n = 3) (not shown). Incubation of SC58125 with these
cells indicated 50-60% recovery of Cox-1 activity in ASA-treated
cells after this prolonged incubation. This is in agreement with a
slower turnover of Cox-1 protein compared to Cox-2 found previously
using [
S]methionine-labeled cells(17) .
Figure 7:
Recovery of the capacity of endothelial
cells to generate TX from ASA-treated platelets after irreversible
inhibition of cyclooxygenase by ASA. Panel A, HUVEC in 12-well
dishes (2 10
cells) were incubated for 30 min in
the absence or presence of ASA or SA (100 µM each). After
6 h activation with IL-1
(25 units/ml), the supernatant was
removed and ASA-treated platelets (PL/EC = 125) were added and
stimulated by thrombin (2 units/ml) for 2 min. Panel B, same
as in A, but cells were incubated for 3 h in the presence of
NHS alone (left panel) or PMA (20 nM). The specific
Cox-2 inhibitor, SC58125 (5 µM), was preincubated 10 min
prior to thrombin stimulation. The results are the mean ± S.E.
of three to four experiments performed under the same
conditions.
We have shown that induction of Cox-2 in human endothelial
cells contributes considerably to the synthesis of PGH which can be metabolized by ASA-treated platelets via
transcellular biosynthesis. Demonstration of substrate transfer for the
synthesis of TXA
is supported by several arguments: (i)
after stimulation of endothelial cells with thrombin, only early
addition of supernatant to ASA-treated platelets generates TX; (ii) an
unstable intermediate is present in the supernatant of stimulated
endothelial cells with an apparent half-life compatible with that of
PGH
; (iii) TX formation is reduced when an antibody
directed against a PGH
mimic is added to the cell
co-incubate; (iv) TX synthesis was inhibited in the presence of a TX
synthase inhibitor; (v)
H-labeled TX is formed when using
[
H]arachidonic acid-labeled endothelial cells in
the presence of aspirin-treated platelets. The release from the cell of
a transient metabolic intermediate, transformed by vicinal acceptor
cells, has been reported in other situations(27) . Results in Fig. 2A suggest that PGH
released in the
supernatant of stimulated cells is available for conversion into TX.
Although a report claimed that only unidirectional transfer of
substrate from platelets to endothelial cells could occur(28) ,
these results are not supported by our studies. This discrepancy could
be explained either by low levels of cyclooxygenase in these cells
and/or by the selective presence of one isozyme.
The discovery that endothelial cells, like other nucleated cells, contain a primary response gene responsible for the expression of Cox-2 upon cell activation calls for careful evaluation of such reactions. Increased presence of Cox-2 has been associated with enhanced capacity to generate prostacyclin(9, 10, 23) . In the present experiments, induction of Cox-2 also correlates with the greater capacity of the cell co-incubate to generate TX (Fig. 4).
An important aspect of the studies reported here
concerns the respective contributions of endothelial cell Cox-1 and
Cox-2 to the ``input'' or ``output'' of substrates (i.e. arachidonic acid and PGH, respectively). The
irreversible inhibition of PG synthesis by the 30-min treatment of
endothelial cells with ASA is rapidly overcome after induction of Cox-2
by different stimuli. It has been suggested that ASA or other
non-steroidal anti-inflammatory drugs may influence de novo synthesis of Cox(29, 30) . Although this could be
due to inhibition of the transcription factor NF-
B by
ASA(31) , concentrations were 10-50 times higher (>1
mM) than those used here. Western blot analysis (Fig. 5) shows that pretreatment of cells with ASA or SA does
not interfere with de novo synthesis of Cox-2. In addition,
the rapid (2-6 h) recovery in activity is due to induction of
Cox-2: (i) in cell lysates of ASA-treated cells, enzyme activity is
only present in the immunoprecipitates of Cox-2 but not of Cox-1; (ii)
in intact cells, the specific Cox-2 inhibitor, SC58125 suppresses
totally the activity in ASA-treated cells stimulated by PMA or
IL-1
but not that of untreated cells; (iii) Cox-1 activity is
partially recovered after longer periods of stimulation (>18 h). In
fact, non-activated HUVEC contain a significant amount of Cox-2 (Fig. 5) which contributes approximately 40-60% of basal
activity. These results are consistent with our previous report (17) of the presence of Cox-2 in control cells maintained in 5%
NHS. After activation of cells and induction of Cox-2, cyclooxygenase
activity is increased 6-8-fold compared to that contributed by
Cox-1 (Fig. 7B). It is, however, difficult to establish
a direct relation between mass of enzymes (estimated in relative
Western blot units, using different antibodies specific for Cox-1 and
Cox-2) and products, since the ``quantitative'' scale for
both parameters (and between each isozyme) is different. Overall, the
biosynthetic capacity of HUVEC to produce PGs appears to be related to
the quantity of enzyme(s) present in the cells.
Some reports have
recently questioned the role of Cox-2 in augmenting constitutive PG
synthesis provided by Cox-1(6, 13) . Enzyme
compartmentalization and the possibility that different intracellular
sources of arachidonic acid restrict each isozyme to a unique pool of
substrate may contribute to a tight regulation of
products(12, 32, 33) . We have investigated
the role of Cox-1 by using control cells in the presence of SC58125
(where Cox-2 is inhibited) or Cox-2 in ASA-treated cells activated for
3-6 h with PMA or IL-1. Exogenous arachidonic acid,
Ca
ionophore A23187, and thrombin were used to
analyze the influence of exogenous substrate versus endogenous
(provided by receptor-dependent or independent cell stimuli) on the
synthesis of PGs from different isozymes. Our experimental data do not
support the concept that Cox-1 and Cox-2 are enzymes of separate
synthetic pathways, despite their specific intracellular sites in
HUVEC(13) . The fact that metabolite production was similar in
response to all inducers argues against a tight relation between the
presence of Cox-1 and Cox-2 in endothelial cells and a selective
production of PGs related to an exogenous or endogenous source of
substrates. Comparable results were obtained in the presence of
microsomes from aspirin-treated platelets, and the synthesis of
6-keto-PGF
was similar in endothelial cells alone and
in the presence of platelets. We suggest that the selectivity of Cox
isozymes with respect to the source of arachidonic acid and enzyme
compartmentalization may also depend on the biological system (cell
source, species, inducer, etc.) as most other examples have been
obtained on cell
lines(12, 32, 33, 34) . Moreover,
reasons inherent to enzyme activities have been presented to explain a
differential control of PG synthesis by the two isoforms when present
in the same cell (35) .
Another aspect of these studies
concerns the release of PGH into the external milieu for
its conversion into TXA
by platelets. Although Cox-1 or
Cox-2 can contribute to constitutive and transcellular metabolisms, a
different response was observed after treatment of cells with ASA.
PGH
generated from newly synthesized Cox-2 produced a
relative increase in intracellular 6-keto-PGF
over
transcellular TXB
(Table 2, ``Cox-2''
column). This suggests that PGH
is more readily
metabolized, by intracellular enzymes at early times of protein
synthesis (i.e. 3 h of induction), than released outside the
cell for transcellular biosynthesis. Short-term synthesis of Cox-2
coincides with location of the enzyme around the nucleus(13) .
These findings reconcile data from Fig. 6and 7 since, after 2 h
activation, aspirin-treated cells recovered >80% of Cox-2 activity
evaluated by 6-keto-PGF
but only 35-50% based on
TXB
production. After 18 h, 90% of 6-keto-PGF
and TXB
synthesis had been recovered.
In summary, in endothelial cells, enzyme compartmentalization occurring after short-term activation may selectively affect transcellular metabolism but not constitutive production of PGs. The quantity of PGs is clearly related to the amount of Cox (mainly type 2) present in cells under our conditions. Because Cox-2 expression can be regulated rapidly and significantly, its level can significantly affect the capacity of endothelial cells to generate PGs. In addition, when Cox-1 is irreversibly inactivated, induction of Cox-2 provides cells with a rapid de novo capacity to generate PGs.