© 1998 American Heart Association, Inc.
Original Contributions |
Differential Induction of Cyclooxygenase-2 in Human Arterial and Venous Smooth Muscle
Role of Endogenous Prostanoids
Correspondence to Jane A. Mitchell, Unit of Critical Care Medicine, Royal Brompton Hospital, Sydney Street, London SW3 6NP, UK. E-mail j.mitchell{at}rbh.nthames.nhs.uk
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Abstract |
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Abstract—Two isoforms of cyclooxygenase (COX) have been identified: a constitutive isoform (COX-1), found in abundance in platelets and the vascular endothelium, and an "inflammatory" cytokine-inducible isoform (COX-2). Because COX metabolites regulate vascular smooth muscle cell (SMC) function and the interaction between the vessel and circulating components, we have investigated the possibility that COX-2 can be induced in human arterial or venous SMC. Untreated venous or arterial cells contained undetectable levels of COX-1 or COX-2 and released low levels of metabolites. After stimulation with interleukin-1ß, tumor necrosis factor-
, interferon-
, and bacterial lipopolysaccharide,
both venous and arterial SMC expressed COX-2 protein and
released increased amounts of prostaglandins. In addition,
the induced release of PGE2 was inhibited by the
COX-2–selective inhibitor, L-745,337. When cells were
treated with the mixture of cytokines, venous SMC expressed
greater amounts of COX-2 protein and released more
prostaglandins than arterial SMC. Furthermore,
when COX-2 activity was blocked by L-745,337, COX-2 expression in
arterial SMC, but not in venous SMC, increased. Thus, this
article describes, for the first time, that COX-2 is expressed in
greater amounts in venous SMC than in arterial SMC.
Moreover, we show that this "differential induction" is due to a
negative-feedback pathway for COX-2 expression in arterial
SMC but not in venous SMC. The ability of COX-2 activity to limit COX-2
expression in some cells but not others may contribute to the highly
developed mechanisms involved in prostanoid release.
Key Words: saphenous vein • internal mammary artery • atherosclerosis • coronary artery bypass grafting
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Introduction |
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Cyclooxygenase (COX)1 is the initial enzyme in the conversion of arachidonic acid to metabolites including prostacyclin (PGI2), TXA2, and PGE2, which have a variety of biological actions involved in the maintenance of vascular homeostasis. PGI2 is released mainly by endothelial cells,2 acts on IP receptors linked to the activation of adenylyl cyclase,3 inhibits platelet aggregation, causes vasodilatation,2 reduces cholesterol accumulation,4 and inhibits vascular smooth muscle cell (SMC) proliferation.5 TXA2 is released mainly by platelets, acts on TP receptors linked to the inositol phosphate pathway, causes vasoconstriction, and stimulates platelet aggregation.3 PGE2 is released by leukocytes as well as endothelial cells and acts on a variety of EP receptors linked to different transduction pathways; therefore, it can produce different vascular responses, depending on the distribution of particular EP receptor subtypes.3
In healthy vessels, endothelial cells are enriched with COX activity,2 whereas the underlying smooth muscle releases considerably lower amounts of prostanoids.6 Under such conditions, the release of COX metabolites by endothelium is regulated by a constitutively expressed isoform of COX (COX-1)7 . Thus, when the endothelium is damaged, COX-1 activity and the release of PGI2 are reduced, thereby rendering the vessel susceptible to vasospasm, thrombosis, and atherosclerosis.
Recently, a cytokine- or mitogen-induced isoform of COX was
identified (COX-2) in chick8 and
murine9 fibroblasts and was demonstrated
consequently in a variety of human cell types including
endothelial cells,10
monocytes,11 airway epithelial
cells,12 and airway smooth
muscle13 in vitro. In addition, COX-2 also has
been demonstrated at the site of inflammation in
vivo.14 15 Agents that induce COX-2 include
interleukin-1ß (IL-1ß), tumor necrosis factor- (TNF-),
bacterial lipopolysaccharide (LPS), growth factors, and phorbol
esters.16 Furthermore, COX-2 is induced in rat
vascular SMC treated with platelet-derived growth factor or
transforming growth factor-ß in vitro and in rat vessels after
physical trauma in vivo.17 We recently have shown
that COX-2 is induced in intact segments of human saphenous vein (SV)
and internal mammary artery (IMA) in organ
culture.18 However, in these studies, the
cellular origin of COX-2 was not determined. Considering that vascular
smooth muscle is a major component of the vascular tree, it is
important to establish the role of COX-2 in prostanoid release by these
cells under physiological and
pathophysiological conditions. Moreover, we have
found that human veins express significantly more COX-2 activity after
induction than human arteries.18 Whether this
differential expression is due to fundamental differences in the
ability of venous and arterial SMC to express COX-2 is not
clear. Thus, in the current report, we have compared the ability of
human arterial and venous SMC in culture to release COX
products and express COX-2 protein in response to a variety of
inflammatory stimuli. Furthermore, we have investigated any possible
feedback mechanisms that prostanoids may have on COX-2 induction in the
2 cell types.
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Methods |
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Materials
Human recombinant IL-1ß and TNF-
were from
Boehringer-Mannheim; IFN- was from R&D Systems; tritiated
prostanoids were from Amersham; all cell culture media and supplements
were supplied by Gibco BRL; L-745,337
[5-methanesulfonamido-6-(2,4-difluorothiophenyl)-1-indanone] and
selective COX-1 and COX-2 antibodies were a gift from Merck Frosst; all
other reagents and antibodies were from Sigma Chemical Co.
Human Vessels
Segments of undistended SV (n=5) and IMA (n=6) were obtained
from patients undergoing coronary artery bypass surgery; 4 of
these IMAs and SVs were obtained from the same patients. Ethical
permission was obtained from the Ethical Committee of the Royal
Brompton National Heart and Lung Hospital. All vessels were used
regardless of preoperative drug therapy or patient history.
Vessel Collection and Cell Culture
All vessels were placed immediately into sterile pots containing
sterile PBS (Pen-Strep) supplemented with penicillin (100 IU/mL) and
streptomycin (0.1 mg/mL; Pen-Strep) and prepared immediately or stored
at 5°C for no more than 2 hours before preparation. Vascular SMCs
were grown by explant outgrowth method, in DMEM containing 1
mmol/L sodium pyruvate and phenol red, supplemented with Pen-Strep,
2 mmol/L glutamine DMEM, and 20% FBS (37°C, 5%
CO2, 95% air). SMCs were identified by
characteristic morphological "hill-and-valley" growth pattern and
by smooth muscle -actin immunostaining.
COX Activity in Human Arterial and Venous Smooth
Muscle
COX activity, supported by either endogenous or
exogenous sources of arachidonic acid, was measured in
cells (only passages 2 to 6 were used) cultured on 96-well tissue
culture plates. COX activity utilizing endogenous stores of
arachidonic acid was measured by the release of
prostanoids in the supernatant of cells treated with different
combinations of drugs for 6-, 12-, 24-, or 48-hour time periods. The
prostanoid release in these experiments relies on the activity of both
phospholipases (eg, A2) to liberate
membrane-bound arachidonic acid and COX. In experiments
designed to asses COX activity directly (ie, without the contribution
of phospholipases), cells were treated with the relevant
cytokines/drugs for the designated times (as described above),
and the culture medium was replaced for 15 minutes with fresh medium
containing arachidonic acid (30 µmol/L). The
agents used to study COX induction were IL-1ß (10 ng/mL), TNF- (10
ng/mL), LPS (Escherichia coli; serotype 0111:B4; 10
µg/mL), or IFN- (1000 U/mL; 24 and 48 hours only), or a
"cytokines mix" consisting of IL-1ß, TNF-, LPS, and
IFN-. In separate experiments, SMCs were treated with the
COX-1/COX-2 inhibitor
indomethacin7 19 or its
COX-2–selective derivative L-745,33720 (10
pmol/L to 100 µmol/L for both) 30 minutes before addition of the
cytokine mixture for an additional 24 hours. The medium was
then removed, and prostanoids were measured by radioimmunoassay. Stocks
of indomethacin or L-745,337 (100 mmol/L) were
dissolved in dimethyl sulfoxide and diluted in DMEM. All nonsterile
aqueous solutions were filtered (0.2 µm) before addition to the
cells.
Western Blot Analysis for COX-1 and COX-2
SV or IMA SMCs were seeded on 6-well plates and left untreated
or treated with the mixture of cytokines for 24 hours. In some
experiments, L-745,337 (10 µmol/L) was also added to the cells.
The medium was then removed, cells were washed with PBS, and proteins
were extracted with a Tris buffer (50 mmol/L; pH 7.4) containing
10 mmol/L EDTA, 1% vol/vol Triton-X 100, and 1 mmol/L PMSF.
Extracts were boiled at a 1:1 ratio with Tris (50 mmol/L; pH 6.8;
4% wt/vol SDS; 10% vol/vol glycerol; 4% vol/vol 2-mercaptoethanol; 2
mg/mL bromophenol blue). Samples of equal protein were loaded onto
7.5% Tris-glycine SDS gels and were separated by
electrophoresis.21 After transfer to
nitrocellulose, the blots were primed with either a selective
anti-human COX-1 or a specific anti-human COX-2
antibody20 (Merk Frosst) raised in rabbit. The
blots were then incubated with anti-rabbit IgG (raised in donkey),
conjugated to horseradish peroxidase, and developed by ECL (Amersham
International Ltd). Rainbow markers (14 000 to 200 000 kDa; Amersham
International Ltd) were used for molecular weight determinations.
Prostanoid Determination
PGE2 was used as the main index of COX
activity because it was the predominant PG released. 6-Keto
PGF1 (the hydrolysis product of
PGI2), TXB2 (the hydrolysis
product of TXA2), and
PGE2 were measured by radioimmunoassay using
commercial antibodies and tritiated prostanoids, as previously
described.7 Antibodies to 6-keto
PGF1 or TXB2 did not
cross-react with disparate prostanoids. However,
PGE2 displayed 
15% cross-reactivity with
the antibody to 6-keto
PGF1.22 Statistics were
calculated from Prism 2.01 (Graphpad Software) using the appropriate
recommended test (see appropriate figure legends).
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Results |
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Effect of IL-1ß, TNF-
, IFN-, or LPS on COX Activity in
Cultures of Human SV and IMA SMCUnstimulated venous and arterial SMC released 12±3 and 13±2 ng/mL PGE2, respectively, over 48 hours. Either IL-1ß (10 ng/mL; Figure 1A
) or TNF- (10 ng/mL; Figure 2A) caused a time-dependent release of
PGE2 from endogenous stores of
arachidonic acid by both venous and
arterial SMC. Similarly, COX activity, supported by
exogenous arachidonic acid, was increased in both
venous and arterial cells by IL-1ß (Figure 1B) or TNF-
(Figure 2B). LPS (Figure 3) significantly
increased the release of PGE2 and COX activity in
venous but not arterial cells. Venous cells released
consistently more PGE2 than
arterial cells when stimulated with IL-1ß (Figure 1). In
contrast, venous and arterial cells released similar
amounts of PGE2 (from either
endogenous or exogenous stores of
arachidonic acid) after stimulation with either TNF-
or LPS (Figures 2 and 3). INF- (1000 U/mL) had no significant effect
on COX activity in either cell type (data not shown). Stimulants had
the following rank order of efficiency as inducers of COX activity:
IL-1ß>TNF->LPS>>INF- (Figures 1, 2, and 3).
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Effect of a Combination of IL-1ß, TNF-, LPS, and INF- on
COX Activity in Human Arterial and Venous SMC
A mixture of IL-1ß (10 ng/mL), TNF- (10 ng/mL), IFN- (1000
U/mL), and LPS (10 µg/mL) produced an "additive" effect on
COX activity from arterial SMC but acted in synergy in
venous SMC (Figure 4). Indeed, the
maximum release of PGE2 in response to the
"cytokine mixture" by venous SMC (Figure 4) was
5-fold greater than the sum of the release by individual
cytokines (Figures 1
, 2, and 3). In separate experiments (n=9),
cells treated with the cytokine mixture released mainly
PGE2 (for arterial cells, 77±5
ng/mL; for venous cells, 846±107) but also increased levels of
6-keto-PGF1 (for arterial cells,
19±1 ng/mL; for venous cells, 84±4 ng/mL), but not levels of
TXB2 (<0.1 ng/mL for arterial or
venous cells) after 48 hours.
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Induction of COX Protein in SV and IMA
COX-1 protein could not be detected in extracts of either
arterial or venous cells cultured in the presence or
absence of cytokines (data not shown). Similarly, under control
culture conditions, no protein for COX-2 was detected in extracts of
arterial or venous SMC (Figure 5). However, when either
arterial or venous SMCs were stimulated for 24 hours with
the mixture of cytokines, a band at 70 kDa was
recognized by the specific COX-2 antibody (Figure 5). Furthermore, a
second band of 60 kDa20 was induced along
with the COX-2 protein after cytokine treatment. A protein
of 40 kDa was also recognized by the COX-2 antibody, although
the expression of this protein did not change with cytokines
(data not shown). In keeping with the differences in COX activity, the
amount of COX-2 protein induced by the cytokine mixture
was 3-fold higher in venous SMC cultures than in
arterial SMC cultures (0.24±0.02 OD units for
arterial cells; 0.75±0.2 OD units for venous cells). When
COX-2 activity was blocked with L-745,337 (10 µmol/L), the COX-2
expressed in cytokine-treated arterial SMC
increased (plus cytokines, 0.26 OD units; plus
cytokines plus L-745,337, 0.6±0.2 OD units; Figure 6), and L745337 did not affect COX-2
expression in venous cells (plus cytokines, 0.7±0.2 OD units;
plus cytokines plus L-745,337, 0.5±0.25 OD units; Figure 7). L-745,337 had no effect on the amount
of COX-2 protein in unstimulated cells (data not shown). In contrast to
its effects on COX-2 expression, L-745,337 inhibited the level of the
coinduced 60-kDa protein by 89±5% in venous SMC and by 72±13% in
arterial SMC (n=3; Figures 6 and 7). In separate
experiments, PGE2 (1 µmol/L) but not
cicaprost (1 µmol/L) inhibited the expression of COX-2 in
cytokine-treated arterial SMCs (Figure 8; control, 0.3±0.1 OD units; plus
PGE2, 0.09±0.05 OD units; n=4) but not in venous
SMCs (control, 0.87±.09 OD units; plus PGE2,
0.78±0.2 OD units; n=4).
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Effects of the COX-2–Selective Inhibitor L-745,337 and
the COX-1/COX-2 Inhibitor Indomethacin on
PGE2 Release by Arterial and Venous
SMC
The inhibitor L-745,337 is approximately equipotent
with indomethacin as an inhibitor of
purified COX-2 and >500 times less potent than
indomethacin as an inhibitor of
COX-1.20 Thus, where L-745,337 is equipotent with
indomethacin, COX-2 is likely to predominate. In our
experiments, both L-745,337 and indomethacin caused
concentration-dependent reductions in PGE2
released by either venous or arterial SMC stimulated by the
cytokine mixture. In cytokine-treated
arterial cells, L-745,337 and indomethacin
were equipotent with pIC50 values of 8.8 and 8.2,
respectively. Similarly, L-745,337 and indomethacin
were approximately equipotent inhibitors of COX activity in
cytokine-activated venous cells with
pIC50 values of 7.6 and 7.8 (Figure 9).
|
) and L-745,337 (
) as
inhibitors of PGE2 release by
cytokine-stimulated venous (A) and arterial (B)
SMC. Data represent mean±SE from 3 patients (each performed in
triplicate). Data are calculated as the percentage of the
PGE2 release occurring in "cytokine
mix"–stimulated cells in the absence of either
indomethacin or L-745,337. |
Discussion |
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Arachidonic acid metabolites formed by the COX pathway have potent actions on vascular smooth muscle contractility,2 3 6 growth,3 5 23 and cholesterol uptake.4 Within vessels, the majority of COX present is in the constitutive form, COX-1, which is expressed mainly in the endothelial layer, with much lower levels being present in the underlying smooth muscle.6 However, data from animal models have suggested that when the endothelium is compromised or vessels are damaged, the inducible isoform, COX-2, can be expressed in vascular smooth muscle.17 Here, we show that COX activity can be increased greatly in human vascular SMC by different cytokines and that, under optimum conditions, venous SMCs have the capacity to release much higher levels of prostanoids than arterial SMC.
Both venous and arterial SMCs released low levels of
COX metabolites when cultured in the absence of cytokines. This
observation is consistent with the notion that SMCs do not
normally express COX enzymes. However, after treatment with either
TNF-, LPS, IL-1ß, or "cytokine mix," both
arterial and venous SMCs release increased amounts of COX
metabolites. Of the metabolites measured, PGE2
was released preferentially followed by PGI2
(measured as its breakdown product,
6-keto-PGF1), with very low levels of
TXB2. This pattern of release is
consistent with our18 24 and
other25 previous findings, confirming the
presence of PGI2 synthetase in
arterial and venous SMC25 26 27 and
that only low levels of TXA2 synthetase are
present. The large amounts of PGE2 released
may be due to saturation of the PGI2 synthetase
pathway and PGH2 being converted
nonenzymatically28 to PGE2,
although a specific link between COX-2 and increasing
PGE2 isomerase activity has been suggested
recently.29
Venous and arterial cells released similar amounts of
PGE2 under "basal conditions" or when
stimulated to release greater amounts with TNF-. However, when cells
were stimulated with IL-1ß or the mixture of cytokines,
venous cells released greater amounts of PGE2
than did arterial cells. Similarly,
cytokine-stimulated venous cells expressed significantly more
COX-2 protein than did arterial cells. In contrast to
bovine aortic endothelium30 or
rat aortic vascular SMCs31 in culture, human
venous and arterial vascular SMCs were relatively
insensitive to LPS as an inducer of COX-2. As freshly isolated human SV
or IMA release PGs readily to LPS,32 this
insensitivity may be the result of long-term culture of human SMC.
The nonsteroidal anti-inflammatory drug indomethacin is a potent nonselective inhibitor of COX-1 and COX-212 19 ; recently, a COX-2–selective derivative, L-745,337, has been described.20 L-745,337 is equipotent with indomethacin as an inhibitor of COX-2 but >500 times less potent as an inhibitor of COX-1. We found indomethacin and L-745,337 to be approximately equipotent inhibitors of the cytokine-induced release of PGE2 by both venous and arterial cells. This observation is again consistent with COX-2 being the active isoform present in our SMC cultures. Interestingly, both indomethacin and L-745,337 were more potent inhibitors of COX in arterial cells than in venous cells. Because arterial cells express less COX-2 than venous cells, nonsteroidal anti-inflammatory drugs may have easier access to protein and thereby be more potent. Indeed, preliminary data obtained by us, using pure COX-2 protein, show that the relative potency of indomethacin increases with lower amounts of enzyme (J.A.M., unpublished data, 1998).
Why, then, do venous cells express more COX-2 protein and activity than equally treated arterial cells? One possible explanation could be that products formed by the COX pathway limit the induction of COX-2 in arterial cells but not in venous cells. In cytokine-treated cells, when COX-2 activity was blocked with the selective inhibitor L-745,337, the expression of COX-2 protein in arterial but not in venous cells was increased. Moreover, COX-2 expression in arterial cells was strongly inhibited by exogenously applied PGE2. This observation is consistent with a recently published study38 ; using porcine aortic SMC, the authors showed that exogenous PGE2 suppresses the induction of COX-2 stimulated by either U46619 (a thromboxane mimetic) or basic fibroblast growth factor. Thus, our data suggest that a "feedback" system exists to limit COX-2 induction in some SMC. Interestingly, the IP receptor agonist, cicaprost, did not affect the expression of COX-2 in either cell type. These observations suggest that COX-2 expression is modulated by EP receptor activation in these arterial cells. Indeed, the differences observed between arterial and venous cells may reflect variations in the prostanoid receptor population expressed on the 2 types of smooth muscle.33 34
Similar to the predicted 70-kDa COX-2 protein, both
arterial and venous SMC expressed a protein of 60
kDa. Similar observations have been made in other cells expressing
COX-2 and in purified protein,20 where the 60-kDa
protein was thought to be a breakdown product of COX-2. We found
that the 60-kDa protein was inhibited by L-745,337 in both
arterial and venous cells but that COX-2 protein was
increased only in the arterial cells. Thus, the 70-kDa
COX-2 and the unidentified 60-kDa protein appear to be regulated
independently. However, the inhibitory effect of L-745,337
on the expression of the 60-kDa protein was reversed by exogenous
PGE2 (Figure 8). This observation suggests that
the 60-kDa protein, like COX-2 expression, is also regulated by
PGE2 release.
The venous and arterial cells used in our study were derived from SV and IMA, 2 commonly used bypass conduits. The artery is the vessel of first choice because it has a lower incidence of reocclusion than the vein. However, with the common need for multiple grafts, and because the artery is not always suitable for grafting,35 SV is still commonly used. The primary reason for graft failure is reocclusion due to atherosclerosis, a process that involves smooth muscle proliferation, inflammatory cell invasion, and lipid accumulation.36 COX-2 products released by cells in this study were mainly PGE2 and PGI2, which are thought to be antilipidemic via both cAMP-dependent activation of cytosolic neutral cholesteryl ester hydrolase and independent activities on other cholesteryl ester cycle enzymes.4 Thus, the induction of COX-2 in IMA and SV SMC may act to reduce cholesterol uptake and metabolism. Furthermore, the release of PGs after COX-2 induction could also inhibit the activity and adhesion of platelets2 and may play a feedback role on the release of cytokines such as IL-1ß, TNF, and CSF-1 (macrophage CSF).4 Thus, induction of COX-2 in vascular SMC may represent a compensatory mechanism that ensures the release of PGI2 and PGE2 when endothelium is compromised. However, COX-2 induction also may have detrimental effects on vascular function. For example, the peroxidase activity of COX can oxidize lipids,37 producing potent atherogenic stimuli. Furthermore, COX activity in human SV results in vasoconstriction, whereas COX activity in human IMA results in vasodilation.33 Thus, the consequences of COX-2 expression in vascular smooth muscle may differ, depending on the type and origin of the vessel.
We have demonstrated recently, using an organ culture technique, that COX-2 is induced in segments of SV to a greater level than in IMA.18 Here, we have extended these studies and identified vascular smooth muscle as an important site of COX-2 induction. Moreover, we have established, similarly to observations in intact tissue,18 that venous cells (even at passage 6) are capable of expressing much greater amounts of COX-2 activity than similarly cultured arterial cells. Furthermore, in arterial cells, but not in venous cells, COX-2 products negatively regulate the expression of COX-2, thereby explaining why arterial cells express less COX-2. These observations add to our current understanding of COX-2 regulation1 16 and establish human vascular smooth muscle as an important site for COX-2 expression. As COX-2 in human vascular SMC is regulated by cytokines found in several vascular diseases, we hypothesize that COX-2 will be expressed rapidly as a response to vascular injury and is likely therefore to play a role in modulating and controlling subsequent vascular pathologies.
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Acknowledgments |
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This work was supported by grants from the Wellcome Trust (J.A.M.) and the British Heart Foundation (FS/94075 to D.B.-B.).
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Footnotes |
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Department of Applied Pharmacology, The National Heart and Lung Institute, Imperial College of Science and Technology (D.B.-B., S.W.L.), London; and Department of Cardiothoracic Surgery (J.R.P.), Unit of Critical Care Medicine (J.A.M.), The Royal Brompton Hospital, London, UK.
Received November 26, 1997; accepted April 6, 1998.
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