Abstract

Objective: Inflammation contributes to atherosclerotic plaque initiation and progression. Recent studies suggest that anti-inflammatory drugs such as cyclooxygenase-2 (Cox-2) inhibitors have anti-atherogenic effects. The current study was designed to investigate whether administration of a Cox-2 inhibitor to older apolipoprotein E deficient (apo E−/−) mice with established lesions alters the composition and increases the stability of the lesions. Methods and results: The Cox-2 inhibitor Celecoxib was administered in chow to 26-week-old, male, apo E−/− mice exhibiting advanced, unstable atherosclerotic lesions within the innominate/brachiocephalic artery. Mice administered Celecoxib had no significant changes in serum cholesterol or the average cross sectional area of atherosclerotic lesion in the innominate artery after 15 weeks of treatment in comparison to non-treated control mice. Histological analyses of sections of the innominate artery demonstrated no significant changes in the frequency of markers of advanced and unstable atherosclerotic plaques, including intra-plaque hemorrhage, vascular calcification, thinning of the fibrous cap, size of the necrotic core and macrophage content. There were also no significant differences in the content of Cox-2 within the lesions. Quantitative real time polymerase chain reaction with mRNA isolated from the aorta of each mouse revealed no significant changes in the expression of tissue factor and inducible nitric oxide synthase. However, mRNA levels for MCP-1 were increased fivefold following 15 weeks of treatment with Celecoxib in comparison to non-treated control mice. Conclusions: These data suggest that Celecoxib has no effect on the composition of advanced atherosclerotic lesions in older apo E−/− mice.

1. Introduction

The rupture of vulnerable atherosclerotic plaques leads to acute coronary events and stroke [1,2]. The vulnerable plaque is generally composed of an atrophic fibrous cap, a lipid-rich necrotic core, dystrophic mineralization, and the accumulation of inflammatory cells such as macrophages and T-cells [3,4]. Macrophages are capable of degrading extra-cellular matrix by secreting active proteolytic enzymes. This could lead to weakening of the fibrous cap with plaque rupture and intra-plaque hemorrhage, and ultimately formation of occlusive thrombi [5–7]. Macrophage-derived cyclooxygenases (Cox) play a key role in inflammation by converting arachidonic acid into prostaglandins and thromboxanes in response to a large number of pro-inflammatory stimuli [8,9].

Cyclooxygenases exist in several different isoforms. Cox-1 is constitutively expressed in various tissues, including the stomach, kidney and platelets [8]. Recently, a new variant of Cox-1 has been identified that is inhibited by acetaminophen and now referred to as Cox-3 [10]. In contrast, Cox-2 is induced in response to growth factors and cytokines such as interleukin-1β, tumor necrosis factor-α and interferon-γ, suggesting a role for Cox-2 in inflammation and cell growth [8,11]. Cox-2 is expressed in human atherosclerotic lesions [12,13] and there is elevated expression in symptomatic versus asymptomatic plaques, linking Cox-2 to plaque destabilization [14]. These observations have led to the hypothesis that selective Cox-2 inhibition might have beneficial effects on reducing vascular inflammation with subsequent plaque stabilization. However, clinical studies have so far failed to demonstrate any improvement in cardiovascular risk in patients treated with Cox-2 inhibitors [15,16]. In fact, clinical studies such as the VIGOR trial have raised concerns that selective Cox-2 inhibition may actually increase the risk of cardiovascular events by decreasing vasodilatory and anti-aggregatory prostacyclin production [17]. In contrast, in a recent study by Burleigh et al., treatment of LDL receptor deficient mice with a selective Cox-2 inhibitor for 6 weeks reduced the macrophage content and size of early atherosclerotic lesions [18]. The effects of Cox-2 inhibition on plaque stability were not determined in this model of early atherosclerotic lesions development.

We and others have recently shown that older apo E−/− mice develop advanced, unstable atherosclerotic lesions in the brachiocephalic/innominate arteries. These lesions have some of the morphologic features of advanced atherosclerotic lesions in humans, including intra-plaque hemorrhage and calcification [19,20] making this a useful model for investigating interventions that may alter plaque composition and increase plaque stability [21]. In the present study, we investigated the effects of selective Cox-2 inhibition on plaque composition in older apo E−/− mice. Lesion progression, changes in morphologic features related to plaque instability, and inflammatory cell content were evaluated. Furthermore, the effects of inhibition of Cox-2 on the aortic expression of tissue factor (TF), monocyte chemoattractant protein-1 (MCP-1), and inducible nitric oxide synthase (iNOS) were investigated, since these pro-inflammatory factors play important roles in the atherogenic process.

2. Methods

2.1. Animals

Male apo E−/− mice on a C57BL/6J background (n = 35) were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Mice were fed a chow diet and water ad libitum throughout the study. At 26 weeks of age, Celecoxib (75 mg/kg/day) was added to the chow of the treatment group (n = 16), while the control group continued to receive regular chow (n = 19). This study was approved by the University of Washington Institutional Animal Care and Use Committee.

2.2. Animal sacrifice and preparation of arterial tissues

Following 15 weeks of Celecoxib treatment, the mice were sedated (Avertin; Aldrich, Milwaukee, WI, USA), blood was collected from the inferior vena cava, and the animals were sacrificed by exsanguination. The animals were perfused with 10 ml ice cold phosphate-buffered saline (PBS) at physiological pressure via the left ventricle. A suture was tied around each aorta distal to the branch of the left subclavian artery. The aortas were collected beginning from the suture and extending to the bifurcation of the femoral arteries and the surrounding fat and connective tissue were removed. The dissected tissues were immediately snap frozen in liquid nitrogen and stored at −80°C until further use. The animals were then perfused with 10% buffered formalin via the left ventricle for 4 min. The brachiocephalic artery from each animal (referred to as the innominate artery) was dissected out, embedded in paraffin and serially sectioned (5 μm). Every fifth section was stained with a modified Movat's pentachrome stain [22]. To identify vascular calcification, adjacent slides were stained with the von Kossa stain [23]. Total serum cholesterol was measured colorimetrically using a commercially available cholesterol oxidase enzymatic kit.

2.3. Real time reverse transcriptase polymerase chain reaction (RT-PCR)

Total RNA was extracted from each aorta into Tripure reagent (Boehringer Mannheim, Mannheim, Germany). Reverse transcription was performed with 1 μg total RNA isolated from each separate aorta. To determine the levels of expression of TF, MCP-1, iNOS and β-actin, real time PCR quantification (TaqMan) was performed (ABI Prism 7700 Sequence Detection System; PE Biosystems, Foster City, CA, USA). The primer sequences for TF, MCP-1, iNOS and β-actin were as follows. TF forward: 5′-CACTCATCATTGTGGGAGCAGTG-3′ and reverse: 5′-CGCGACGGGGTGTTCTT-3′; for MCP-1 forward: 5′-TCTGGGCCTGCTGTTCAC-3′ and reverse: GTGAATGAGTAGCAGCAGGTGAGT-3′, for iNOS forward: 5′-CAGCAGCGGCTCCATGAC-3′ and reverse: 5′-CGGCACCCAAACACCAAG-3′ and for β-actin forward: 5′-CCCTAAGGCCAACCGTGAAA-3′ and reverse 5′-ACGACCAGAGGCATACAGGGA-3′. The sequences for the probes were TF: 5′-CATATCTCTGTGCAAGCGC-3′, MCP-1: 5′-CTCAGCCAGATGCAGTTAA-3′, iNOS: 5′-TCATGCGGCCTCCTTTGAGCCCT-3′ and for β-actin: 5′-ATGACCCAGATCATGTTTGAGACCTTCAACAC-3′. The transcript for the constitutive gene product β-actin was used for data normalization.

2.4. Immunohistochemistry

Tissue sections of the innominate artery (5 μm) were stained with an anti-mouse macrophage antibody (Mac-3; Pharmingen, San Diego, CA, USA), anti-smooth muscle actin antibody (1A4; Dako, Carpenteria, CA, USA) and anti-Cox-2 (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturers’ protocols. The extent and distribution of the reaction products within the lesions were determined using computer-assisted morphometry (Image Pro; Media Cybernetics, Silver Spring, MD, USA).

2.5. Evaluation of plaque composition and plaque size

Two independent investigators blinded to the study protocol, evaluated each section for the frequency of features characteristic of plaque instability. These included: thickness of the fibrous cap (thin fibrous cap was defined as three or fewer cell layers), size of the necrotic core (a large necrotic core was defined as occupying more than 50% of the volume of the plaque), intra-plaque hemorrhage (defined as the presence of red blood cells independent of microvessels), calcification (defined on the basis of positive staining with the von Kossa stain), medial erosion (defined as enlargement of the media caused by infiltration of plaque components) and lateral xanthomas (defined as the presence of aggregates of macrophage-derived foam cells situated on the lateral margins of the plaques). These were recorded as binary outcomes and the frequency for each animal was determined. The cross sectional area of lesion in each section was determined using computer-assisted morphometry (Image Pro; Media Cybernetics) and is reported as maximum plaque area per animal.

2.6. Statistical analyses

All data were expressed as mean±S.E. Significant differences between means in serum cholesterol and lesion size were determined with the Student's two-tailed t-test. For analysis of gene expression and plaque morphology, groups were compared using the Mann–Whitney U-test.

3. Results

3.1. Effects of Celecoxib on drug levels, serum cholesterol and maximum lesion area

Treatment with Celecoxib (75 mg/kg/day) led to a serum drug level of 537.6±185.1 ng/ml (Table 1). There were no significant differences in serum cholesterol levels between groups following 15 weeks of the drug treatment (Table 1). Furthermore, no significant differences were observed in the average maximum cross sectional area of atherosclerotic lesions in the innmominate arteries of the treated animals versus controls (Table 1).

Table 1

Drug levels, serum cholesterol and average cross sectional lesion area in Celecoxib-treated and control apo E−/− mice

Treatment Drug level Total cholesterol Lesion area 
 (ng/ml)* (mg/dl)* (μm2)* 
15 weeks Celecoxib 537.6±185.1 455.8±83.2 145,848±56,148 
15 weeks control n.d. 499.7±96.9 140,357±51,897 
Treatment Drug level Total cholesterol Lesion area 
 (ng/ml)* (mg/dl)* (μm2)* 
15 weeks Celecoxib 537.6±185.1 455.8±83.2 145,848±56,148 
15 weeks control n.d. 499.7±96.9 140,357±51,897 

* Values shown are the means±S.E.

n.d.=Non detectable.

3.2. Effects of Celecoxib on the aortic expression of tissue factor, MCP-1 and iNOS

Real time RT-PCR was performed with total mRNA isolated from the aortas of each of the apo E−/− mice. Following 15 weeks of Celecoxib treatment, there were no significant changes in expression of TF or iNOS (Fig. 1). However, a significant increase was observed in expression of MCP-1 in the aortas of the Celecoxib-treated mice relative to control mice (approximate 5.2-fold increase, P<0.05, Fig. 1).

Fig. 1

Effects of Celecoxib on aortic gene expression in apo E−/− mice. (A) Relative mRNA abundance of tissue factor, monocyte chemoattractant protein-1 and inducible nitric oxide synthase in the aorta of Celecoxib-treated and control mice measured by real time PCR. All data were normalized to β-actin expression. Values shown are the means±S.E. (*=P<0.05).

Fig. 1

Effects of Celecoxib on aortic gene expression in apo E−/− mice. (A) Relative mRNA abundance of tissue factor, monocyte chemoattractant protein-1 and inducible nitric oxide synthase in the aorta of Celecoxib-treated and control mice measured by real time PCR. All data were normalized to β-actin expression. Values shown are the means±S.E. (*=P<0.05).

3.3. Effects of Celecoxib on plaque composition

No significant differences in plaque composition were observed following 15 weeks of treatment with Celecoxib (Fig. 2). There were no significant differences in the frequencies of lateral xanthomas and intra-plaque hemorrhage. Hemorrhage was most frequently observed within the necrotic zones, in the lateral xanthomas and underneath thin fibrous caps in the lateral margins of the lesions in all of the animals (Fig. 3, panels A&B). There were also no significant differences in the frequencies of intra-plaque calcification, thin fibrous caps (Fig. 3, panel C), medial erosion, size of the necrotic cores or presence of perivascular inflammation (Fig. 3, panel D). Immunostaining for macrophages also demonstrated no significant differences in the macrophage content in the innominate arteries of the Celecoxib-treated and control mice (13±3.9% of lesion area in the control animals versus 19.2±5.2% in the Celecoxib-treated animals). In both the treated and control mice, macrophages were predominantly located in lateral xanthomas and diffusely distributed throughout the lipid-rich necrotic cores (data not shown). Cox-2 was expressed primarily by macrophages situated adjacent to the necrotic core of the lesions, in the lateral margins of the lesions as well as along the lumen of the lesions (Fig. 4). Again, there were no significant differences in the percentage of the lesion area immunostained with an antibody for Cox-2 within the lesions of the treated and control mice (23.75±7.9% of lesion area for the control animals versus 17.5±2.9% for the treated animals). No significant changes in smooth muscle cell content were observed.

Fig. 4

Immunohistochemistry of advanced lesions in the innominate artery of apo E−/− mice. Sections showing positive staining for cyclooxygenase-2 in control (panel A), and 15 weeks Celecoxib-treated (panel B) apo E−/− mice. Lateral margins of lesions (panel A, closed arrows) as well as cells within the necrotic core (panel A, open arrows) showing positive staining for cyclooxygenase-2. Areas within the luminal cell layers (panel B, closed arrows) as well as areas within the necrotic core (panel B, open arrows) demonstrating staining for cyclooxygenase-2. Bar=100 μm.

Fig. 4

Immunohistochemistry of advanced lesions in the innominate artery of apo E−/− mice. Sections showing positive staining for cyclooxygenase-2 in control (panel A), and 15 weeks Celecoxib-treated (panel B) apo E−/− mice. Lateral margins of lesions (panel A, closed arrows) as well as cells within the necrotic core (panel A, open arrows) showing positive staining for cyclooxygenase-2. Areas within the luminal cell layers (panel B, closed arrows) as well as areas within the necrotic core (panel B, open arrows) demonstrating staining for cyclooxygenase-2. Bar=100 μm.

Fig. 3

Morphology of advanced lesions in the innominate artery of apo E−/− mice. Movat's pentachrome stained sections showing intra-plaque hemorrhage in the central core (panel A, closed arrow), and in the lateral margins (panel B, closed arrow) of atherosclerotic lesions in the innominate artery of control (panel A) and Celecoxib-treated animals (panel B). Note the thin fibrous cap overlaying a necrotic core with intra-plaque hemorrhage in the control animals (panel A, open arrow) versus the thicker fibrous cap in the Celecoxib-treated animal (panel B, open arrow). (C) Advanced lesion of the innomiante artery of a control animal demonstrating extensive areas of intra-plaque calcification. (D) Movat's pentachrome stained section of a Celecoxib-treated animal demonstrating medial erosion (closed arrow) and the formation of a lateral xanthoma (open arrow). L=Lumen, Calc.=calcification. Bar=100 μm.

Fig. 3

Morphology of advanced lesions in the innominate artery of apo E−/− mice. Movat's pentachrome stained sections showing intra-plaque hemorrhage in the central core (panel A, closed arrow), and in the lateral margins (panel B, closed arrow) of atherosclerotic lesions in the innominate artery of control (panel A) and Celecoxib-treated animals (panel B). Note the thin fibrous cap overlaying a necrotic core with intra-plaque hemorrhage in the control animals (panel A, open arrow) versus the thicker fibrous cap in the Celecoxib-treated animal (panel B, open arrow). (C) Advanced lesion of the innomiante artery of a control animal demonstrating extensive areas of intra-plaque calcification. (D) Movat's pentachrome stained section of a Celecoxib-treated animal demonstrating medial erosion (closed arrow) and the formation of a lateral xanthoma (open arrow). L=Lumen, Calc.=calcification. Bar=100 μm.

Fig. 2

Frequency of morphologic markers of plaque stability in the innominate artery of Celecoxib-treated and control apo E−/− mice. Values shown are the means±S.E.

Fig. 2

Frequency of morphologic markers of plaque stability in the innominate artery of Celecoxib-treated and control apo E−/− mice. Values shown are the means±S.E.

4. Discussion

The results of the present study suggest that chronic administration of Celecoxib to older apo E−/− mice does not inhibit the progression of advanced lesions in the innominate artery or alter the composition of the plaques. These data are consistent with the observations of Duffy et al. [24], who found that treatment with a selective Cox-2 inhibitor, SC-236, also had no effect on lesion development in younger apo E−/− mice. Furthermore, in a study from the same group inhibition of Cox-2 in a rat balloon angioplasty model had no effect on intimal hyperplasia [25]. In contrast, Burleigh et al. reported that treatment of LDL receptor deficient mice with Rofecoxib inhibited lesion progression [18]. The differences between the present observations and those of other groups may be related to the differences in mouse strain, to differences in the age of the animals and to differences in the site of the lesions evaluated and composition of the lesions at the time of administration of the Cox-2 inhibitors. It is likely that the anti-inflammatory effects of the drugs may be more evident in early lesions composed predominantly of leukocytes.

In comparison to early lesions at all vascular sites, there are reduced numbers of macrophages within the advanced lesions in the innominate arteries of the apo E−/− mice. In the older mice, intact macrophages are located predominantly within small lateral xanthomas [26]. Furthermore, lesions in the innominate arteries of the older apo E−/− mice do not appreciably increase in size after about 36–42 weeks of age and this may also help explain the failure of the Celecoxib to inhibit progression in the present study [26]. The differences in the effects of Cox-2 inhibitors on early versus established lesions may also be due to the progressive buildup and release of oxidation products in the advanced lesions. Eligini et al., have recently reported that oxidized phospholipids inhibit expression of Cox-2 in macrophages [27]. Thus, Cox-2 expression may have already been reduced independently of the inhibitor and this would explain the similar levels of Cox-2 protein observed within the plaques of the treated and control mice and the failure of the inhibitor to alter the properties of the lesions.

The primary focus of the present study was to determine whether the Cox-2 inhibitor altered plaque composition and contributed to the stability of advanced lesions. The data clearly indicate that Celecoxib does not have any significant effects on altering the composition or stability of advanced lesions in the apo E−/− mice. One must be very cautious in equating these observations with mouse models of advanced atherosclerosis with the potential effects of Cox-2 inhibitors on advanced human atherosclerosis. However, as noted, recent human trials such as the VIGOR study suggest that Cox-2 inhibitors may also not alter advanced plaque composition in humans [17].

TF, MCP-1 and iNOS are key factors involved in the atherogenic process. It is likely that MCP-1 promotes the infiltration of monocytes into the vascular wall and their transformation into macrophage-derived foam cells at all stages of lesion development [28,29]. TF is the initiating factor in the extrinsic pathway of coagulation, can be activated by a variety of inflammatory stimuli [30,31], and appears to contribute to immediate thrombus formation after plaque disruption [32]. Production of nitric oxide by iNOS expressed by macrophages and smooth muscle cells has an important bactericidal function but may also contribute to oxidant stress. It has also been shown to reduce the collagen content in advanced lesions and thus, could contribute to plaque instability [33,34].

The data from the current study demonstrate that chronic Celecoxib treatment of older apo E−/− mice does not reduce the aortic expression of these pro-inflammatory genes. In fact, expression of MCP-1 in the aorta was increased after treatment with the Cox-2 inhibitor. This may be due to the fact that cyclooxygenase inhibitors have been shown to attenuate the production of prostaglandin E2 (PGE2), a potent suppressor of MCP-1 expression both in vivo and in vitro [35–38]. In a recent study by Takayama et al. using cDNA microarrays, PGE2 inhibited lipopolysaccharide induced expression of pro-inflammatory cytokines, such as MCP-1 and interleukin-8 in human monocytes. Treatment of the cells with a selective Cox-2 inhibitor suppressed PGE2 production and restored the production of the pro-inflammatory cytokines [39]. It is unclear why the increased expression of MCP-1 in the treated mice did not lead to an increase in the macrophage content of the lesions in the innominate arteries. However, hypercholesterolemia itself is a potent inducer of macrophage recruitment and there are additional mechanisms that are redundant with MCP-1 that may have accounted for our failure to observe an additive effect of the increased MCP-1 expression. It is also possible, although unlikely, that the increase in MCP-1 mRNA did not translate into an increase in MCP-1 protein.

We did not observe a significant difference in the content of Cox-2 within the lesions, despite the relatively high dosage of Celecoxib and sufficient plasma levels of the drug. In contrast, Burleigh et al., found reduced expression of Cox-2 in early lesions of young LDL receptor deficient mice after treatment with Rofecoxib [18]. Differences in the strain and age of the mice as well as use of a different Cox-2 inhibitor may again account for the differences between these studies. However, as noted, a buildup of oxidation products in the advanced lesions as compared to early lesions may have already reduced expression of Cox-2 [27]. It is also feasible that the capacity of the inhibitor to block Cox-2 expression was overwhelmed by the stimulatory effects of the many pro-inflammatory factors released into the necrotic zones of the advanced lesions. These include; IL-1, TNF-alpha, MIF-1, HSP-60, ceramide, peroxynitrite, and proteases, all previously shown to stimulate Cox-2 expression in vitro [40–44].

The current paradigm for the formation of occlusive thrombi and subsequent coronary events and stroke involves rupture of the fibrous cap, intra-plaque hemorrhage, and exposure of blood to thrombogenic plaque components including the lipid-rich necrotic core, inflammatory cells, and/or vascular calcification [45]. In this regard, there is evidence that suggests that products of the cyclooxygenases may promote expression of matrix metalloproteinases (MMPs) in macrophages [14,46]. The proteolytic activity of these enzymes may contribute to plaque instability in advanced atherosclerotic plaques [47]. In the current study however, Cox-2 inhibition resulted in no change in the frequency of intra-plaque hemorrhage suggesting that inhibition of cyclooxygenase 2 does not attenuate the proteolytic function of the MMPs in this model or that increased expression and activation of MMPs may not play a significant role in stimulating plaque rupture and fissures in the innominate arteries of older apo E−/− mice.

5. Conclusions

This is the first report of the effects of the inhibition of cyclooxygenase-2 on atherosclerotic plaque composition and plaque stability in older apo E−/− mice. Celecoxib had no effects on plaque composition in this mouse model of advanced atherosclerotic lesions suggesting that chronic inhibition of cyclooxygenase-2 may not have an anti-inflammatory effect on advanced atherosclerotic lesions.

Acknowledgements

We would like to thank Jerry Ricks, Carl P. Storey, Elizabeth P. Wallace, Monica I. Shelley and Annette Buttler for expert technical assistance. We would also like to thank Daniel Donnelly, Brad Keller, and Elaine Krul for help in the design and implementation of the project and for critical feedback. This study was supported by a non-restricted grant from Pharmacia, Inc. (now a division of Pfizer, Inc.) and by NIH HL 58954.

References

[1]
Fuster
V.
Lewis
A.
Conner Memorial Lecture. Mechanisms leading to myocardial infarction: insights from studies of vascular biology
Circulation
 
1994
90
2126
2146
[2]
Hansson
G.K.
Immune mechanisms in atherosclerosis
Arterioscler Thromb Vasc Biol.
 
2001
21
1876
1890
[3]
Libby
P.
Molecular bases of the acute coronary syndromes
Circulation
 
1995
91
2844
2850
[4]
Schmermund
A.
Erbel
R.
Unstable coronary plaque and its relation to coronary calcium
Circulation
 
2001
104
1682
1687
[5]
Hatsukami
T.S.
Ross
R.
Polissar
N.L.
Yuan
C.
Visualization of fibrous cap thickness and rupture in human atherosclerotic carotid plaque in vivo with high-resolution magnetic resonance imaging
Circulation
 
2000
102
959
964
[6]
Bini
A.
Mann
K.G.
Kudryk
B.J.
Schoen
F.J.
Noncollagenous bone matrix proteins, calcification, and thrombosis in carotid artery atherosclerosis
Arterioscler Thromb Vasc Biol.
 
1999
19
1852
1861
[7]
Burke
A.P.
Kolodgie
F.D.
Farb
A.
Weber
D.
Virmani
R.
Morphological predictors of arterial remodeling in coronary atherosclerosis
Circulation
 
2002
105
297
303
[8]
Dubois
R.N.
Abramson
S.B.
Crofford
L.
et al
Cyclooxygenase in biology and disease
FASEB J.
 
1998
12
1063
1073
[9]
Hinson
R.M.
Williams
J.A.
Shacter
E.
Elevated interleukin 6 is induced by prostaglandin E2 in a murine model of inflammation: possible role of cyclooxygenase-2
Proc Natl Acad Sci USA
 
1996
93
4885
4890
[10]
Chandrasekharan
N.V.
Dai
H.
Roos
K.L.
et al
From the cover: COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression
Proc Natl Acad Sci USA
 
2002
99
13926
13931
[11]
Simon
L.S.
Role and regulation of cyclooxygenase-2 during inflammation
Am J Med.
 
1999
106
37S
42S
[12]
Schonbeck
U.
Sukhova
G.K.
Graber
P.
Coulter
S.
Libby
P.
Augmented expression of cyclooxygenase-2 in human atherosclerotic lesions
Am J Pathol.
 
1999
155
1281
1291
[13]
Baker
C.S.
Hall
R.J.
Evans
T.J.
et al
Cyclooxygenase-2 is widely expressed in atherosclerotic lesions affecting native and transplanted human coronary arteries and colocalizes with inducible nitric oxide synthase and nitrotyrosine particularly in macrophages
Arterioscler Thromb Vasc Biol.
 
1999
19
646
655
[14]
Cipollone
F.
Prontera
C.
Pini
B.
et al
Overexpression of functionally coupled cyclooxygenase-2 and prostaglandin E synthase in symptomatic atherosclerotic plaques as a basis of prostaglandin E(2)-dependent plaque instability
Circulation
 
2001
104
921
927
[15]
Mukherjee
D.
Nissen
S.E.
Topol
E.J.
Risk of cardiovascular events associated with selective COX-2 inhibitors
J. Am. Med. Assoc.
 
2001
286
954
959
[16]
Pitt
B.
Pepine
C.
Willerson
J.T.
Cyclooxygenase-2 inhibition and cardiovascular events
Circulation
 
2002
106
167
169
[17]
Bombardier
C.
Laine
L.
Reicin
A.
et al
Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis. VIGOR Study Group
New Engl J Med.
 
2000
343
1520
1528
[18]
Burleigh
M.E.
Babaev
V.R.
Oates
J.A.
et al
Cyclooxygenase-2 promotes early atherosclerotic lesion formation in LDL receptor-deficient mice
Circulation
 
2002
105
1816
1823
[19]
Rosenfeld
M.E.
Polinsky
P.
Virmani
R.
et al
Advanced atherosclerotic lesions in the innominate artery of the apoE knockout mouse
Arterioscler Thromb Vasc Biol.
 
2000
20
2587
2592
[20]
Williams
H.
Johnson
J.L.
Carson
K.G.
Jackson
C.L.
Characteristics of intact and ruptured atherosclerotic plaques in brachiocephalic arteries of apolipoprotein E knockout mice
Arterioscler Thromb Vasc Biol.
 
2002
22
788
792
[21]
Bea
F.
Blessing
E.
Bennett
B.
et al
Simvastatin promotes atherosclerotic plaque stability in ApoE-deficient mice independently of lipid lowering
Arterioscler Thromb Vasc Biol.
 
2002
22
1832
1837
[22]
Movat
H.Z.
Demonstration of all connective tissue components in a single section
Arch Pathol.
 
1955
60
289
295
[23]
Mallory
F.B.
Pathological techniques, 2nd ed.
1942
Philadelphia, PA
W.B. Saunders
[24]
Duffy
A.
Fitzgerald
D.J.
Belton
O.A.
Inhibition of cyclooxygenase isoforms and the development of atherosclerosis in the APOE knockout mouse
Circulation
 
2001
104
II-64
Abstract
[25]
Connolly
E.
Bouchier-Hayes
D.J.
Kaye
E.
et al
Cyclooxygenase isozyme expression and intimal hyperplasia in a rat model of balloon angioplasty
J Pharmacol Exp Ther.
 
2002
300
393
398
[26]
Rosenfeld
M.
Kauser
K.
Martin-McNulty
B.
et al
Estrogen inhibits the initiation of fatty streaks throughout the vasculature but does not inhibit intra-plaque hemorrhage and the progression of established lesions in apolipoprotein E deficient mice
Atherosclerosis
 
2002
164
251
259
[27]
Eligini
S.
Brambilla
M.
Banfi
C.
et al
Oxidized phospholipids inhibit cyclooxygenase-2 in human macrophages via nuclear factor-kappaB/IkappaB- and ERK2-dependent mechanisms
Cardiovasc Res.
 
2002
55
406
415
[28]
Boring
L.
Gosling
J.
Cleary
M.
Charo
I.F.
Decreased lesion formation in CCR2−/− mice reveals a role for chemokines in the initiation of atherosclerosis
Nature
 
1998
394
894
897
[29]
Aiello
R.J.
Bourassa
P.A.
Lindsey
S.
et al
Monocyte chemoattractant protein-1 accelerates atherosclerosis in apolipoprotein E-deficient mice
Arterioscler Thromb Vasc Biol.
 
1999
19
1518
1525
[30]
Guha
M.
O'Connell
M.A.
Pawlinski
R.
et al
Lipopolysaccharide activation of the MEK-ERK1/2 pathway in human monocytic cells mediates tissue factor and tumor necrosis factor alpha expression by inducing Elk-1 phosphorylation and Egr-1 expression
Blood
 
2001
98
1429
1439
[31]
Bavendiek
U.
Libby
P.
Kilbride
M.
et al
Induction of tissue factor expression in human endothelial cells via CD40 ligand is mediated by AP-1, NF-κB, and Egr-1
J Biol Chem.
 
2002
277
25032
25039
[32]
Toschi
V.
Gallo
R.
Lettino
M.
et al
Tissue factor modulates the thrombogenicity of human atherosclerotic plaques
Circulation
 
1997
95
594
599
[33]
Niu
X.L.
Yang
X.
Hoshiai
K.
et al
Inducible nitric oxide synthase deficiency does not affect the susceptibility of mice to atherosclerosis but increases collagen content in lesions
Circulation
 
2001
103
1115
1120
[34]
Detmers
P.A.
Hernandez
M.
Mudgett
J.
et al
Deficiency in inducible nitric oxide synthase results in reduced atherosclerosis in apolipoprotein E-deficient mice
J Immunol.
 
2000
165
3430
3435
[35]
Jocks
T.
Zahner
G.
Freudenberg
J.
et al
Prostaglandin E1 reduces the glomerular mRNA expression of monocyte-chemoattractant protein 1 in anti-thymocyte antibody-induced glomerular injury
J Am Soc Nephrol.
 
1996
7
897
905
[36]
Schneider
A.
Harendza
S.
Zahner
G.
et al
Cyclooxygenase metabolites mediate glomerular monocyte chemoattractant protein-1 formation and monocyte recruitment in experimental glomerulonephritis
Kidney Int.
 
1999
55
430
441
[37]
Martin
C.A.
Dorf
M.E.
Differential regulation of interleukin-6, macrophage inflammatory protein-1, and JE/MCP-1 cytokine expression in macrophage cell lines
Cell Immunol.
 
1991
135
245
258
[38]
Peebles
R.S.
Jr.
Dworski
R.
Collins
R.D.
et al
Cyclooxygenase inhibition increases interleukin 5 and interleukin 13 production and airway hyperresponsiveness in allergic mice
Am J Respir Crit Care Med.
 
2000
162
676
681
[39]
Takayama
K.
Garcia-Cardena
G.
Sukhova
G.K.
et al
Prostaglandin E2 suppresses chemokine production in human macrophages through the EP4 receptor
J Biol Chem.
 
2002
277
44147
44154
[40]
Wu
D.
Marko
M.
Claycombe
K.
et al.  
Paulson
K.E.
Meydani
S.N.
Ceramide-induced and age-associated increase in macrophage COX-2 expression is mediated through up-regulation of NF-kappa B activity
J Biol Chem.
 
2003
278
10983
10992
[41]
Billack
B.
Heck
D.E.
Mariano
T.M.
et al
Induction of cyclooxygenase-2 by heat shock protein 60 in macrophages and endothelial cells
Am J Physiol Cell Physiol.
 
2002
283
C1267
C1277
[42]
Houliston
R.A.
Keogh
R.J.
Sugden
D.
et al
Protease-activated receptors upregulate cyclooxygenase-2 expression in human endothelial cells
Thromb Haemost.
 
2002
88
321
328
[43]
Ahmad
N.
Chen
L.C.
Gordon
M.A.
Laskin
J.D.
Laskin
D.L.
Regulation of cyclooxygenase-2 by nitric oxide in activated hepatic macrophages during acute endotoxemia
J Leukoc Biol.
 
2002
71
1005
1011
[44]
Mitchell
R.A.
Liao
H.
Chesney
J.
et al
Macrophage migration inhibitory factor (MIF) sustains macrophage proinflammatory function by inhibiting p53: regulatory role in the innate immune response
Proc Natl Acad Sci USA
 
2002
99
345
350
[45]
Davies
M.J.
Going from immutable to mutable atherosclerotic plaques
Am J Cardiol.
 
2001
88
2F
9F
[46]
Miralles
M.
Wester
W.
Sicard
G.A.
Thompson
R.
Reilly
J.M.
Indomethacin inhibits expansion of experimental aortic aneurysms via inhibition of the cox2 isoform of cyclooxygenase
J Vasc Surg.
 
1999
29
884
892
[47]
Shankavaram
U.T.
Lai
W.C.
Netzel-Arnett
S.
et al
Monocyte membrane type 1-matrix metalloproteinase. Prostaglandin-dependent regulation and role in metalloproteinase-2 activation
J Biol Chem.
 
2001
276
19027
19032

Author notes

Time for primary review 14 days.