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*
Rheumatology Unit, Royal Adelaide Hospital, and
Division of Human Immunology, Hanson Centre for Cancer Research, Adelaide, South Australia, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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caused induction of COX-2. TXA2 was the
predominant COX-1-derived product, and TXA2 synthesis
changed little with up-regulation of COX-2 by IL-1 (2-fold
increase). By contrast, COX-2 up-regulation was associated with large
increases in the synthesis of PGI2 and PGE2
(54- and 84-fold increases, respectively). Addition of the selective
COX-2 inhibitor, NS-398, almost completely abolished PGI2
and PGE2 synthesis, but had little effect on
TXA2 synthesis. The up-regulation of COX-2 by IL-1 was
accompanied by specific up-regulation of PGI synthase and PGE synthase,
but not TX synthase. An examination of the substrate concentration
dependencies showed that the pathway of TXA2 synthesis was
saturated at a 20-fold lower arachidonic acid concentration than that
for PGI2 and PGE2 synthesis. In conclusion,
endothelial prostanoid synthesis appears to be differentially regulated
by the induction of COX-2. The apparent PGI2 and
PGE2 linkage with COX-2 activity may be explained by a
temporal increase in total COX activity, together with selective
up-regulation of PGI synthase and PGE synthase, and different kinetic
characteristics of the terminal synthases. These findings have
particular importance with regard to the potential for cardiovascular
consequences of COX-2 inhibition. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PGI2 and TXA2 are products of arachidonic acid (AA) metabolism by cyclooxygenase (COX), followed by metabolism of the COX product, PGH2, by the terminal synthase enzymes, prostacyclin or TX synthase, respectively. Two isoforms of COX have been identified: COX-1 is expressed constitutively in most cell types, whereas COX-2 is induced by inflammatory stimuli such as bacterial endotoxin and cytokines. Also, several different prostanoid terminal synthases can be present within the one cell, and it is not known what determines the relative rate of production of each individual prostanoid within the same cell. In monocytic cells, it has been observed that the ratio of PGE2/TXA2 produced is not fixed, but varies according to which COX isoform is present. For example, in rat peritoneal macrophages, under conditions in which only COX-1 was expressed, TXA2 was synthesized in excess of PGE2. However, under conditions of stimulation in which COX-2 was induced, the profile of prostanoid production shifted to favor PGE2 over TXA2 production (4, 5).
It is considered that PGI2 is the main prostanoid synthesized by vascular endothelium and TXA2 is the main prostanoid produced by platelets. However, the endothelium has been reported to synthesize TXA2 in addition to PGI2 (6), and both COX isoforms have been observed, with only COX-1 being detectable in unstimulated cells (6, 7). Endothelial COX-2 can be up-regulated in vitro by inflammatory stimuli (6, 8) and shear stress (7, 9). Because the balance between PGI2 and TXA2 production is central in the maintenance of vascular tone and platelet aggregation, determination of the roles of endothelial COX isozymes, particularly with regard to the contribution of COX-2 in the regulation of prostanoid biosynthesis by the endothelium, is important.
In the current study, we examined the synthesis of prostanoids derived
from either COX-1 or COX-2 by HUVECs. We observed that
TXA2 is the predominant COX-1 product, whereas
up-regulation of COX-2 by IL-1 is associated with a greater increase
in the synthesis of PGI2 and
PGE2 than TXA2. Both PGI
synthase and PGE synthase, but not TX synthase, were up-regulated by
IL-1. Additionally, an examination of the substrate concentration
dependencies of PGI2, PGE2,
and TXA2 synthesis suggests that different
kinetic parameters of the terminal synthases are a major determinant of
the dominance of PGI2 and
PGE2 production when COX-2 was induced by
IL-1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human rIL-1 was from Genzyme (Cambridge, MA). AA, NS-398
(N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide,
rabbit polyclonal Ab against human COX-2, and murine mAb against COX-1
were all purchased from Cayman Chemicals (Ann Arbor, MI).
Peroxidase-labeled donkey anti-rabbit and goat anti-mouse Abs,
ECL Western blotting system,
[3H]PGE2,
[3H]6-keto PGF1
, and
[3H]TXB2 were purchased
from Amersham Pharmacia Biotech (Piscataway, NJ).
PGH2 and (4-(4-fluorophenyl)-2-(4-methylsulfinyl
phenyl)-5-(4-pyridyl)1H-imidazole) (SB 203580) were obtained
from Calbiochem (San Diego, CA). Abs to the phosphorylated forms of p38
and p44/42 mitogen-activated protein kinases (MAPKs) and the
mitogen-activated protein/extracellular signal-related kinase 1
inhibitor PD98059 were purchased from New England Biolabs (Beverly,
MA). Rabbit anti-serum against PGE2 and
6-keto PGF1 and mouse mAb against
-actin were obtained from Sigma (St. Louis, MO).
Cell culture
HUVECs were isolated as described (10). The cells were cultured on gelatin-coated culture flasks in medium M199 with Earle’s salts supplemented with 20% FCS, 25 µg/ml endothelial growth supplement (Genome Therapeutics, Waltham, MA), and 25 µg/ml heparin. Cells between passages 2 and 4 were plated in 24-well dishes (1.5 x 105/ml) and allowed to reach confluence (24 h).
Cell stimulation
HUVECs were incubated with RPMI 1640 medium (containing 10 mM
HEPES, 2 mM L-glutamine, 100 U/ml penicillin, and 100
µg/ml gentamicin), supplemented with 10% heat-inactivated FCS either
in the presence or absence of IL-1 (1 ng/ml, 37°C). For short-term
stimulation (15 min) with either AA or PGH2,
cells were incubated in serum-free RPMI 1640 medium. To inhibit COX-1
activity, untreated cells were pretreated with aspirin (acetylsalicylic
acid (ASA); 10 µg/ml) for 30 min, followed by two washes
(11), and then incubated in the appropriate medium with
the test agents, according to the specified experiment. Other
inhibitors were added 15 min before stimulation. Following the
appropriate treatment, cell supernatants were collected and stored at
-20°C until analysis for prostanoid measurement by RIA.
Prostanoid measurement
TXB2, 6-keto PGF1
(the stable hydrolysis products of TXA2 and
PGI2, respectively), and
PGE2 were measured by RIA using commercially
available reagents, except for the TXB2
antiserum, which was prepared as described previously
(12).
Western blotting
Cell lysates were prepared by treating cells with ice-cold lysis
buffer (HEPES-buffered HBSS, pH 7.4, 0.5% Triton X-100, 10 µg/ml
leupeptin, 10 µg/ml aprotinin) and sample buffer (0.125 M Trizma
base, pH 6.8, 20% glycerol, 4% SDS, 10% 2-ME), followed by 6 min,
95°C before storing at -20°C. Proteins were separated by 9%
SDS-PAGE and then transferred onto a Sequi-Blot polyvinylidene
difluoride membrane (Bio-Rad, Hercules, CA). After blocking the
membranes with 5% fat-free dried milk in TBS (25 mM Tris-HCl, 0.2 M
NaCl, 0.15% Tween 20, pH 7.6), they were incubated with the
appropriate primary Abs, followed by HRP-conjugated donkey
anti-rabbit or sheep anti-mouse Ab. Equivalent protein loading
and transfer efficiency were verified by staining for -actin. Bound
Abs were revealed with ECL reagent, according to the manufacturer’s
protocol.
Statistical analysis
Results are expressed as mean ± SEM of triplicate incubations. Statistical significance was examined by Student’s t test, using p < 0.05 as the significance level.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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COX-2 was not detectable in unstimulated HUVECs (Fig. 1
) and remained undetectable in the
absence of IL-1 over the 24-h time period examined (data not shown).
Treatment of HUVECs with IL-1 (1 ng/ml) for increasing times
resulted in expression of COX-2, which was maximal by 16 h (Fig. 1). By comparison, COX-1 was expressed in unstimulated cells, and its
expression did not change with IL-1 treatment (Fig. 1). A time
course of prostanoid production by HUVECs in response to IL-1
treatment indicated very different synthesis profiles between
TXA2 and PGI2 or
PGE2. TXA2 synthesis was
evident at the earliest time point of 2 h, when COX-1 was the only
isozyme detectable. As COX-2 was induced, there was a modest increase
in TXA2 production by 
2-fold. Even in the
absence of IL-1 stimulation, TXA2 synthesis
increased with time (albeit less than IL-1-stimulated HUVECs) (Fig. 2A). By comparison, in the
absence of IL-1 stimulation, synthesis of PGI2
or PGE2 was not detectable. Treatment of HUVECs
with IL-1 resulted in little or no PGI2 and
PGE2 synthesis up to 4 h, but with the
induction of COX-2 by IL-1, the production of these prostanoids
increased by 54- and 84-fold, respectively (production at 2 h
compared with that at 24 h) (Fig. 2, B and
C). To determine more specifically the IL-1-induced
changes in synthetic capacity of each prostanoid pathway, HUVECs were
treated with IL-1 at time intervals of 0, 8, and 24 h, washed,
and then stimulated with AA (10 µM, 10 min, 37°C). This type of
examination ensures constant substrate concentration. The results under
these conditions were similar to those obtained with measurement of
prostanoid accumulation, as described above. TXA2
was the predominant prostanoid to be synthesized by untreated HUVECs
(i.e., time 0), with PGI2 and
PGE2 being minor products (Table I). At this time, COX-1 was the only
isozyme detectable (Fig. 1). With the induction of COX-2 by IL-1,
production of TXA2 increased by 2-fold at
20 h, whereas PGI2 and
PGE2 synthesis increased by 31.6- and 39.3-fold,
respectively (Table I). Therefore, it appears that
TXA2 is the major COX-1-derived product, but the
induction of COX-2 results in a preferential increase in
PGI2 and PGE2
synthesis.
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To investigate the contributions of the COX isotypes to
endogenously derived prostanoid synthesis, selective inhibition of
COX-1 and COX-2 activities was required. We have previously documented
that transient pretreatment of unstimulated monocytes with aspirin
(ASA) results in irreversible inhibition of COX-1 activity, with no
effect on activity or induction of COX-2 (11). Inhibition
of COX-1 activity by ASA resulted in significant inhibition of
TXA2 synthesis by IL-1-treated HUVECs (42%),
but had no effect on either PGI2 or
PGE2 production (Table II). By comparison, treatment with
the selective COX-2 inhibitor, NS-398, resulted in almost complete
inhibition of IL-1-induced PGI2 and
PGE2 synthesis (91% and 92.5%, respectively),
but only slightly reduced TXA2 production
(Table II).
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, cells were then
washed and incubated with 10 µM AA for 15 min. In untreated cells,
inhibition of COX-1 activity resulted in significant inhibition of
synthesis of TXA2 (82%), the predominant
COX-1-derived prostanoid synthesized by HUVECs (Fig. 3A). Inhibition of COX-1
activity by ASA pretreatment had no significant effect on the synthesis
of PGI2 or PGE2, but
TXA2 levels were inhibited by 35% (Fig. 3). By
contrast, selective inhibition of COX-2 activity by NS-398 (0.5 µM)
resulted in almost complete inhibition of PGI2
and PGE2 production (Fig. 3, B and
C), but synthesis of TXA2 was
inhibited by only 23% (Fig. 3A). Neither ASA nor NS-398
affected the amount of COX-2 protein expressed in response to IL-1
(Fig. 4). These data suggest that while
the majority of TXA2 synthesis is COX-1
dependent, COX-2 can contribute to the synthesis of
TXA2 by IL-1-treated HUVECs. By comparison,
synthesis of both PGI2 and
PGE2 appears to be predominantly COX-2
derived.
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It is possible that the preferential up-regulation of endothelial
PGI2 and PGE2 synthesis by
IL-1 may have resulted from increased amount or activity of PGI
synthase and PGE synthase, respectively. To examine this possibility,
we studied the effects of IL-1 on the conversion of exogenous
PGH2 to TXA2,
PGI2, and PGE2 by the
respective terminal synthases. Addition of PGH2
(10 µM) to untreated HUVECs resulted in production of all prostanoids
being examined. Treatment of the cells with IL-1 (1 ng/ml) for
20 h, before addition of PGH2, did not alter
the production of TXA2, but synthesis of
PGI2 and PGE2 significantly
increased by 10- and 6-fold, respectively (Fig. 5). These results indicate that treatment
of HUVECs with IL-1 results in increased COX-2 expression and
increased expression of PGI synthase and PGE synthase, but not TX
synthase.
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We have reported recently that the kinetic properties of the
terminal synthases are an important determinant of prostanoid
production in human monocytes (13). Therefore, we examined
the kinetic properties of TX synthase, PGI synthase, and PGE synthase
in HUVECs, in response to increasing substrate (AA) availability.
Addition of exogenous AA at doses up to 5 µM to untreated HUVECs,
i.e., only COX-1 present, resulted in a dose-dependent increase in
TXA2 production. However, no further increases in
TXA2 production were observed with higher doses
of AA (10–100 µM), suggesting saturation of TX synthase or COX-1 at
5 µM AA (Fig. 6A). By
contrast, both PGI2 and
PGE2 synthesis increased dose dependently with
increasing AA concentrations, up to at least 50 µM. This indicates
that COX-1 is not saturated at 5 µM AA (Fig. 6A). The
concentration of AA required to achieve half-maximal stimulation of
TXB2 synthesis was 1.2 µM compared with
PGI2 and PGE2, which were
21.9 and 23.6 µM, respectively. Furthermore, at substrate
concentrations up to 5 µM, the apparent rate constant of TX synthase
was greater than that for PGI or PGE synthase. Similar dose responses
to AA were observed in IL-1-stimulated endothelial cells, in which
COX-2 was induced (Fig. 6B). TXA2
production increased dose dependently at doses of AA up to 10 µM,
after which no further increases in TXA2
production were observed. However, synthesis of
PGI2 and PGE2 increased
dose dependently with increasing AA concentrations, up to at least 50
µM. The concentration of AA required to achieve half-maximal
TXA2 synthesis was 2.6 µM, which was
considerably less than that required for half-maximal production of
PGI2 (8.5 µM) and PGE2
(13.2 µM).
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The MAPK cascade is one of the major signaling pathways leading
from cellular activation to gene transcription. Induction of COX-2 has
been reported to be mediated by both the p38 and p44/42 MAPK pathways
in various cell types, in response to either LPS or cytokine
stimulation (14, 15, 16). Therefore, we examined the effect of
IL-1 addition (1 ng/ml) on p38 and p44/42 activation in HUVECs. As
shown in Fig. 7, IL-1 induced
phosphorylation of both p38 and p44/42 MAPK in a time-dependent manner.
Activation of p38 MAPK peaked at 15 min after exposure to IL-1, and
maximal activation of p44/42 MAPK was observed 30 min post-IL-1
treatment. In the absence of IL-1, there was no detectable
phosphorylation of either p38 or p44/42 MAPK (data not shown).
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can activate both the p38 and p44/42
MAPK pathways in HUVECs, we wanted to establish the potential roles of
these MAPKs in the induction of COX-2 expression by HUVECs in response
to IL-1. Addition of SB 203580 (p38 MAPK inhibitor) or PD 98059
(inhibitor of mitogen-activated protein/extracellular signal-related
kinase 1 activation) had no effect on prostanoid synthesis by untreated
HUVEC (data not shown). However, addition of either SB 203580 or PD
98059 to IL-1-treated HUVECs resulted in significant inhibition of
both PGI2 and PGE2
synthesis (Table III). By comparison,
addition of either of these inhibitors had no effect on
TXA2 production (Table III). Recently, doubts
have been raised concerning the selectivity of the MAPK inhibitors SB
203580 and PD 98059. In particular, they have been reported to
interfere directly with AA metabolism via the COX pathway in platelets
(17). However, we did not observe inhibition of
COX-1-derived prostanoids, namely TXA2,
indicating that at the concentrations used, neither compound inhibits
COX-1 or TX synthase activities. Western blot analysis demonstrated
that both SB 203580 and PD 98059 inhibited IL-1-induced COX-2
induction (Fig. 8). Thus, IL-1 appears
to up-regulate COX-2 expression in endothelial cells through a
mechanism involving both the p38 MAPK and the p44/42 MAPK pathways. The
p38 MAPK pathway has been reported to be involved in the regulation of
COX-2 mRNA stability (16, 18), while the p44/42 MAPK has
been shown to regulate COX-2 at the transcriptional level
(16). Whether this specificity of COX-2 regulation by
these MAPKs occurs in our cell system is yet to be elucidated.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, the
synthesis of PGI2 and PGE2
increased substantially, whereas only a modest increase in
TXA2 production was observed. These differential
changes in prostanoid synthesis were observed when production arising
from either endogenous or exogenous AA was measured. Selective
inhibition of either COX-1 or COX-2 supported the apparent dependencies
of TXA2 synthesis of COX-1 and
PGI2 and PGE2 synthesis on
COX-2 in endothelial cells. Similar associations have been observed in rat peritoneal macrophages. COX-1 was linked with TXA2 production, whereas the induction of COX-2 by LPS shifted prostanoid synthesis to favor PGE2 (5, 19) and PGI2 synthesis (5). Differences in the subcellular distributions of COX-1 and COX-2 were proposed as an explanation for the different prostanoid synthesis profiles associated with the different COX isozymes (5). However, this is unlikely, as COX-1 and COX-2 are reported to be located within the same subcellular locations (20).
Both PGI synthase and PGE synthase have been shown to be inducible
enzymes. Expression of PGI synthase was increased by shear stress in
HUVECs (7), and inflammatory stimuli have been reported to
up-regulate PGE synthase activity in rat peritoneal macrophages and
A549 cell line (19, 21, 22). With regard to PGE synthase,
a cytosolic constitutive and a membrane-associated inducible form have
recently been identified (21, 23). Based on coexpression
studies in transfected HEK293 cells, cytosolic constitutive PGE
synthase and membrane-associated inducible PGE synthase are reported to
be functionally linked with COX-1 or COX-2, respectively (21, 23). In the present study, up-regulation of endothelial COX-2
was accompanied by specific up-regulation of the terminal synthases,
PGI synthase and PGE synthase, but not TX synthase. Although this may
explain in part the selective increase in PGI2
and PGE2 production with cell stimulation, other
factors appear to be involved. Synthesis of PGI2
and PGE2 increased by 50- to 80-fold with
endogenous AA, or 31- to 39-fold with exogenous AA, whereas PGI and PGE
synthase activities increased only by 6- to 10-fold. To account for
the magnitude of the increased ratios of
PGI2/TXA2 and
PGE2/TXA2 with COX-2
induction, we propose that different kinetic characteristics of the
terminal synthases may be involved also. Examination of increasing
concentrations of substrate in either untreated or IL-1-treated
cells demonstrated that at the lower doses of AA (
10 µM),
TXA2 synthesis exceeded that of
PGI2 and PGE2, suggesting
that TX synthase has a higher rate constant than that for PGI and PGE
synthase. However, at doses of AA >10 µM, synthesis of
TXA2 did not increase, whereas synthesis of both
PGI2 and PGE2 increased.
Because production of PGI2 and
PGE2 was responsive to doses of AA >10 µM and
up to 50 µM, this demonstrates that COX was not saturated. Therefore,
the lack of responsiveness of TXA2 synthesis
indicates saturation of TX synthase at AA concentrations <10 µM.
These results are in accordance with the differences in
Km values reported for TX and PGE
synthase in human monocytes, which were 1 and 17 µM, respectively
(13). Consideration of the kinetic characteristics of the
terminal synthases allows an explanation of findings without invoking
linkage of COX isotypes with terminal synthases in different
subcellular locations. Thus, it is proposed that under conditions of
low total COX activity, as observed when COX-1 only is present,
TXA2 production predominates due to a higher rate
constant of TX synthase. Upon cell stimulation, total COX activity
increases due to COX-2 induction, TX synthase becomes rapidly saturated
with PGH2, whereas PGI and PGE synthases respond
to the increased COX activity with increased synthesis of
PGI2 and PGE2. In this
explanation, the linkages between COX-1 and TXA2
synthesis and between COX-2 and
PGI2/PGE2 are apparent
linkages only. Thus, a major determinant of increased
PGI2 and PGE2 production
over TXA2 by IL-1 treatment is increased total
COX activity in combination with different kinetic characteristics of
the terminal synthases. The selective increase in PGI synthase and PGE
synthase activity further augments the increases in
PGI2 and PGE2 synthesis
over that of TXA2.
This study indicates that the role of endothelial COX-2 induction in
vascular homeostasis is important due to its action of altering the
ratio of prostanoids from a prothrombotic (high
TXA2/PGI2) to an
antithrombotic (high
PGI2/TXA2) mixture.
Although the induction of COX-2 appears to be important in many
physiological processes, the induction of COX-2 has generally been
associated with production of deleterious prostanoids due to the
involvement of COX-2 in inflammatory disorders such as rheumatoid
arthritis and osteoarthritis (24). Consequently, there has
been rapid development of selective COX-2 inhibitors. These have been
shown to suppress unwanted inflammation in patients with rheumatoid
arthritis and osteoarthritis, with decreased upper gastrointestinal
side effects compared with conventional agents. The selective COX-2
inhibitors are now in clinical use (24, 25). However,
little is known regarding the physiological role of COX-2 in the
vasculature, and there is evidence to suggest that it may be
cardioprotective (26). COX-2 knockout mice are reported to
develop cardiac fibrosis (27), and administration of COX-2
inhibitors abolished the cardioprotective effect of ischemic
preconditioning in a model of myocardial infarction. In this model,
up-regulation of myocardial COX-2 by ischemia was associated with
PGI2 and PGE2 synthesis
(28). Our study indicates that induction of COX-2 in the
endothelium would result in increased synthesis of
PGI2, but not TXA2,
favoring an antithrombotic state. Therefore, up-regulation of COX-2 by
these cells may represent an important protective mechanism against
vascular injury or insult. This could result in an exacerbation of the
potential for thrombotic complications with the use of selective COX-2
inhibitors. Additionally, COX-2 inhibitors do not suppress
COX-1-derived TXA2 production by platelets,
unlike conventional nonsteroidal anti-inflammatory drugs, and this
would further alter the
TXA2/PGI2 balance toward a
prothrombotic state. Two recent studies examining the effects of
selective COX-2 inhibition in healthy volunteers have demonstrated
inhibition of systemic PGI2 production without
inhibition of platelet-derived TXA2 production
synthesis (29, 30). Systemic PGI2
synthesis is measured by urinary excretion of 2,3 dinor-6 keto
PGF1, and is believed to reflect mainly blood
vessel-derived PGI2 synthesis. COX-2 may be
up-regulated in large straight tracts of vasculature that are exposed
to the biomechanical stimulus of uniform laminar flow (9, 31), and this may account for the COX-2-derived
PGI2 production. However, the situation in vivo
remains unclear, because COX-2 was not detected in healthy arterial and
venous tissues, but was highly expressed in atherosclerotic
lesions (32, 33, 34, 35). The cardiovascular consequences of COX-2
inhibition are further emphasized by a recent large clinical trial in
rheumatoid arthritis that reported a 4-fold increase in myocardial
infarction in patients using a selective COX-2 inhibitor
(36).
In summary, the results of this study indicate a mechanism through which the initial prothrombotic vascular response to injury by endothelial cells becomes self-limiting, through the induction of COX-2 and the increased production of PGI2. The results further suggest that the changing total cellular COX activity in conjunction with the kinetic properties of the terminal prostanoid synthases and the selective induction of PGI synthase, but not TX synthase, mediate this response. Furthermore, preliminary results from specific COX-2 inhibition in healthy volunteers and clinical arthritis trials support the contention that vascular COX-2 is an important protein for maintaining vascular homeostasis.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gillian E. Caughey, Rheumatology Unit, Royal Adelaide Hospital, Adelaide, South Australia 5000, Australia. E-mail address: gillian.caughey{at}adelaide.edu.au
3 Abbreviations used in this paper: PGI2, prostacyclin; TX, thromboxane; AA, arachidonic acid; ASA, acetylsalicylic acid; COX, cyclooxygenase; MAPK, mitogen-activated protein kinase.
Received for publication November 27, 2000. Accepted for publication June 20, 2001.
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S. Eligini, S. Stella Barbieri, V. Cavalca, M. Camera, M. Brambilla, M. De Franceschi, E. Tremoli, and S. Colli Diversity and similarity in signaling events leading to rapid Cox-2 induction by tumor necrosis factor-{alpha} and phorbol ester in human endothelial cells Cardiovasc Res, February 15, 2005; 65(3): 683 - 693. [Abstract] [Full Text] [PDF]
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 F. Miceli, A. Tropea, F. Minici, M. Orlando, G. Lamanna, M. F. Gangale, B. Panetta, F. Tiberi, S. Vaccari, R. Canipari, et al. Effects of Insulin-Like Growth Factor I and II on Prostaglandin Synthesis and Plasminogen Activator Activity in Human Umbilical Vein Endothelial Cells J. Clin. Endocrinol. Metab., January 1, 2005; 90(1): 372 - 378. [Abstract] [Full Text] [PDF]
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D. X. Zhang, K. M. Gauthier, Y. Chawengsub, B. B. Holmes, and W. B. Campbell Cyclooxygenase- and lipoxygenase-dependent relaxation to arachidonic acid in rabbit small mesenteric arteries Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H302 - H309. [Abstract] [Full Text] [PDF]
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G.D. Norata, E. Callegari, H. Inoue, and A.L. Catapano HDL3 Induces Cyclooxygenase-2 Expression and Prostacyclin Release in Human Endothelial Cells Via a p38 MAPK/CRE-Dependent Pathway: Effects on COX-2/PGI-Synthase Coupling Arterioscler Thromb Vasc Biol, May 1, 2004; 24(5): 871 - 877. [Abstract] [Full Text]
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 M J James and L G Cleland Applying a research ethics committee approach to a medical practice controversy: the case of the selective COX-2 inhibitor rofecoxib J. Med. Ethics, April 1, 2004; 30(2): 182 - 184. [Abstract] [Full Text]
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F. Cipollone, B. Rocca, and C. Patrono Cyclooxygenase-2 Expression and Inhibition in Atherothrombosis Arterioscler Thromb Vasc Biol, February 1, 2004; 24(2): 246 - 255. [Abstract] [Full Text]
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S. J Armstrong, Y. Xu, and S. T Davidge Effects of chronic PGHS-2 inhibition on PGHS-dependent vasoconstriction in the aged female rat Cardiovasc Res, February 1, 2004; 61(2): 333 - 338. [Abstract] [Full Text] [PDF]
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 S Battersby, H O D Critchley, A J de Brum-Fernandes, and H N Jabbour Temporal expression and signalling of prostacyclin receptor in the human endometrium across the menstrual cycle Reproduction, January 1, 2004; 127(1): 79 - 86. [Abstract] [Full Text] [PDF]
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G. Wu, A. P. Mannam, J. Wu, S. Kirbis, J.-L. Shie, C. Chen, R. J. Laham, F. W. Sellke, and J. Li Hypoxia induces myocyte-dependent COX-2 regulation in endothelial cells: role of VEGF Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2420 - H2429. [Abstract] [Full Text] [PDF]
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L. M. Title, K. Giddens, M. M. McInerney, M. J. McQueen, and B. A. Nassar Effect of cyclooxygenase-2 inhibition with rofecoxib on endothelial dysfunction and inflammatory markers in patients with coronary artery disease J. Am. Coll. Cardiol., November 19, 2003; 42(10): 1747 - 1753. [Abstract] [Full Text] [PDF]
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E. Tuleja, F. Mejza, A. Cmiel, and A. Szczeklik Effects of Cyclooxygenases Inhibitors on Vasoactive Prostanoids and Thrombin Generation at the Site of Microvascular Injury in Healthy Men Arterioscler Thromb Vasc Biol, June 1, 2003; 23(6): 1111 - 1115. [Abstract] [Full Text] [PDF]
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O. Belton and D. Fitzgerald Cyclooxygenase-2 inhibitors and atherosclerosis J. Am. Coll. Cardiol., May 21, 2003; 41(10): 1820 - 1822. [Full Text] [PDF]
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K. Shinmura, M. Nagai, K. Tamaki, M. Tani, and R. Bolli COX-2-derived prostacyclin mediates opioid-induced late phase of preconditioning in isolated rat hearts Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2534 - H2543. [Abstract] [Full Text] [PDF]
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 R. Maas, E. Schwedhelm, J. Albsmeier, and R. H Boger The pathophysiology of erectile dysfunction related to endothelial dysfunction and mediators of vascular function Vascular Medicine, August 1, 2002; 7(3): 213 - 225. [Abstract] [PDF]
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