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Cardiovascular Research Advance Access first published online on October 24, 2008
This version [Corrected Proof] published online on November 16, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn287
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Selective cyclooxygenase-2 inhibition directly increases human vascular reactivity to norepinephrine during acute inflammation

Nabil Foudi1,2, Liliane Louedec1, Thierry Cachina1, Charles Brink1 and Xavier Norel1,*

1 INSERM U698: Haemostasis, Bio-engineering and Cardiovascular Remodeling, CHU X. Bichat, 46, rue Henri Huchard, Paris 75018, France
2 Paris XIII University, Villetaneuse 93430, France

* Corresponding author. Tel: +33 1 40257529; fax: +33 1 40258602. E-mail address: xnorel{at}hotmail.com

Received 17 April 2008; revised 16 October 2008; accepted 20 October 2008

Time for primary review: 17 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: The use of cyclooxygenase-2 (COX-2) inhibitors has been reported to be associated with detrimental vascular events. The aim of our study was to evaluate the role of COX-2 activity in the control of human vascular tone under inflammatory conditions.

Methods and results: Using organ bath experiments, the contraction induced by norepinephrine (NE), U46619 [GenBank] , acetylcholine, and KCl was performed on isolated human internal mammary arteries (IMA) cultured in the presence or absence of both interleukin-1β (IL-1β) and lipopolysaccharide (LPS) with or without endothelium. Under these conditions the COX (cyclooxygenase) isoforms were detected by immunohistochemistry and western blot, and the prostaglandins (PG) and thromboxane (Tx) released were measured using an enzyme immunoassay kit. A significant decrease in the maximal effect induced by NE but not by other stimuli was observed in the IL-1β- and LPS-treated preparations after 6 and 24 h of culture (–19 ± 6 and –25 ± 4%, respectively), an effect that was endothelium independent. Under this inflammatory condition, the COX-2 inhibitors DFU (1 µmol/L), DuP-697 (0.5 µmol/L), and Etoricoxib (1 µmol/L) markedly restored and increased the vascular reactivity to NE. These alterations were not observed with SC-560 (1 µmol/L), a selective COX-1 inhibitor. In addition, the COX-1 isoform was always detected and the COX-2 isoform was only found in human IMA exposed for 6 or 24 h under inflammatory conditions. The COX-2 induction was accompanied by an increase in PGE2 (prostaglandin E2) and PGI2 (prostaglandin I2) release in the culture medium (~2.5-fold) but not with an increase in TxA2 (thromboxane A2) release.

Conclusion: These observations suggest that the inhibition of COX-2 directly potentiates the human vascular tone induced by NE under inflammatory conditions.

KEYWORDS Vascular tone; COX-2 inhibitors; Human internal mammary arteries; Vasoconstriction; Prostaglandins


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Arachidonic acid is released from membrane phospholipids by phospholipase A2 activity.1,2 A metabolic cascade following arachidonic acid release is initiated by the cyclooxygenases (COX) and leads to the formation of prostanoids [prostaglandins (PG) and thromboxane (Tx)]: PGD2, PGE2, PGF2{alpha}, PGI2, and TxA2 which are synthesized by PGD-, PGE-, PGF-, PGI-, and Tx- Synthases, respectively. These prostanoids play a major role in the control of the human vascular tone.3,4 Two isoforms of the COX are abundantly described in mammalian tissues.5 These isoforms derive from two distinct genes; COX-1 is expressed constitutively while the COX-2 (cyclooxygenase-2) expression is induced specifically under inflammatory conditions.6–10 In humans, an increase in the COX-2 activity is associated with many pathological situations such as arthritis, cancer, and during inflammatory conditions related to atherosclerosis and aneurysm.7,8,11–13

The inhibition of the COX-2 by coxibs such as rofecoxib (VIOXX) has been associated with cardiovascular events.14,15 These effects are probably due to an imbalance between PGI2 and TxA2 synthesis which act respectively as anti- and prothrombotic mediators.16 COX-2 inhibition does not suppress COX-1-derived TxA2 production in platelets, unlike non-selective non-steroidal anti-inflammatory drugs, thereby tipping the TxA2–PGI2 balance towards a prothrombotic state. In the human vessels, PGD2 and PGI2 cause vasodilatation, PGF2{alpha} and TxA2 induce vasoconstriction, while PGE2 may produce both effects.3,17 Generally, healthy vascular walls produce significant levels of PGI2 in comparison with the other prostanoids.3 However under certain non-physiological conditions, the endothelial cells or smooth muscle from human blood vessels can preferentially secrete high levels of PGI2, PGE2 and/or PGD2.6,9,18–21 These effects were observed on cells undergoing shear stress produced by increased blood flow and on cells submitted to either several hours of hypertensive agonists, hypoxic or pro-inflammatory conditions. In these models, the induction of COX-2 has been observed. There are few in vitro studies concerning a physiological role of COX-2.22 No association between the expressions of COX isoforms and their respective roles in the control of the human vascular reactivity have been reported. Therefore, the aim of this study was to examine the effects of COX-2 inhibitors on human vascular tone and to characterize the expression and the activity of the different COX isoforms specifically under inflammatory conditions. Our hypothesis is that during acute inflammation, the COX-2 can be induced and inhibition may directly modify the vascular tone.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
2.1 Human vascular preparations
Human internal mammary arteries (IMA) from patients undergoing a coronary bypass surgery (n = 31; 27 male and 4 female, aged 66 ± 7 years old) were obtained from the cardiovascular service at Bichat Hospital (Paris, France). The investigation conforms to the principles outlined in the Declaration of Helsinki as these tissues were anonymized (rendered non-identifiable). All research programs involving the use of human tissue were approved and supported by the INSERM Ethics Committee and these tissues are considered as surgical waste in accordance with French ethical laws (L.1211-3–L.1211-9).

2.2 Organ culture
The IMA were dissected free from connective tissue and placed immediately into 12-well plates containing RPMI (Gibco, pH 7.4) supplemented with antibiotics (penicillin, 1000 IU/mL; streptomycin, 100 µg/mL) and antimycotic (amphotericin, 0.25 µg/mL) in absence or presence of both interleukin-1β (IL-1β, 10 ng/mL) and lipopolysaccharide (LPS; 100 µg/mL) designated, respectively, as ‘Control’ and ‘inflammatory’ conditions. The volume of the culture medium was adjusted to 1 mL for 70 mg of tissue. All tissue incubations were done at 37°C in a humidified atmosphere of 5% CO2 in air using a culture incubator for either 6 or 24 h. Preparations from each individual patient were used either for the 6 or 24 h protocols. Subsequent to this exposure, four different protocols on each patient sample were made. First, vascular preparations were placed in an organ bath for monitoring the smooth muscle reactivity. The second procedure involved the measurement of prostanoids in culture and organ bath media by enzyme immunoassay kit. Finally, two parts of the same samples were separately placed in either paraformaldehyde 4% for immunohistochemistry or frozen (–20°C) for western blot analysis.

2.3 Organ bath and isometric measurements
IMA preparations, cut as rings of 3 mm width (4–16 rings per IMA), were set up in 10 mL organ baths containing Tyrode’s solution (concentration mmol/L): NaCl 139.2, KCl 2.7, CaCl2 1.8, MgCl2 0.49, NaHCO3 11.9, NaH2PO4 0.4, glucose 5.5, gassed with 5% CO2 and 95% O2 at 37°C and pH 7.4. Care was taken not to disrupt the integrity of the endothelium while the lumen of other rings was rubbed in order to remove the endothelium. Each ring was initially stretched to an optimal load (~1.5 g). Changes in force were recorded by isometric force displacement transducer (Narco F-60) and physiographs (Linseis). Rings were then equilibrated for 90 min with bath fluid changes taking place every 10 min. Initial concentration–response curves were obtained with norepinephrine (NE), acetylcholine (ACh), U46619 [GenBank] an analogue of TxA2 or KCl added in a cumulative fashion to the baths (0.1 nmol/L–10 µmol/L). A precontraction was induced by U46619 [GenBank] (0.1 µmol/L) for ACh relaxation. In the IMA preparations contracted previously by NE, when a maximal effect was obtained, the preparations were washed with Tyrode’s solution until they returned to the resting tone. Subsequently, these preparations were incubated (30 min) with the COX-1-selective inhibitor (SC-560, 1 µmol/L) or a COX-2-selective inhibitor (DFU 1 µmol/L; DuP-697, 0.5 µmol/L or Etoricoxib, 1 µmol/L) and a second concentration–response curve of NE was obtained. Each preparation was used for one protocol (two concentration–response curves of NE separated with an incubation period). In order to compare the viability of preparations cultured after 6 or 24 h with fresh one, IMA samples (0 h) were immediately placed in the organ bath for vascular reactivity measurement to NE as soon as possible after surgery (<2 h).

2.4 Prostaglandin E2, 2,3-dinor-6-keto- prostaglandin F1{alpha} and thromboxane B2 measurements
The supernatants of culture medium in presence or absence of IL-1β and LPS were collected after 6 and 24 h. The concentrations of PGE2, 2,3-dinor-6-keto-PGF1{alpha}, a stable metabolite of PGI2 and TxB2, a stable metabolite of TxA2 were measured in the culture medium using an enzyme immunoassay kit (Cayman) according to the manufacturer’s instructions. In addition, the PGE2 and 2,3-dinor-6-keto-PGF1{alpha} were measured in the organ bath medium after 30 min of incubations with or without a selective COX-2 inhibitor DuP-697 (0.5 µmol/L). The results are expressed as pg/mg of tissue wet weight.

2.5 Immunohistochemistry
Transversal slices (5 µm) of IMA cultured for 0, 6 and 24 h in Control or inflammatory conditions were obtained from paraffin-embedded preparations. Sections were deparaffinized and rehydrated. Endogenous peroxidases were blocked by hydrogen peroxide (Sigma) and antigen retrieval was performed in antigen unmasking solution (Vector). The monoclonal anti-human COX-1 and COX-2 antibodies (Cayman) were used at 1:50 dilution at 4°C overnight. Biotinylated anti-mouse was the secondary antibody and peroxydase Vectastain® Elite ABC kits were used for detection.

2.6 Western blot analysis
Human IMA samples were harvested using a polytron apparatus at 4°C in Tris–HCl buffer: Tris 50 mmol/L pH = 8, NaCl 150 mmol/L, Triton X-100 1%, sodium desoxycholate 1%, SDS 0.1%, EDTA 5 mmol/L. The buffer was added with a protease inhibitor cocktail (Sigma-Aldrich). Proteins were quantified by Lowry’s method. A hundred microgram of protein sample and 100 ng of COX-1 and COX-2 protein recombinant (Cayman), used as standard, were loaded on a 10% polyacrylamide gel. Proteins were blotted onto nitrocellulose membranes (Amersham Biosciences). Membranes were blocked (TBS, 0.1% Tween-20, 5% non-fat dry milk) and incubated overnight at 4°C with a monoclonal anti-COX-1 or anti-COX-2 antibodies (Cayman) diluted at 1:200 in TBS/0.1% Tween-20. Subsequently the membranes were incubated with alkaline phosphatase-conjugated goat anti-mouse secondary antibody (Sigma-Aldrich). Bands were visualized using the electrochemical luminescence plus system (Amersham Biosciences).

2.7 Data analysis
Acquisition and processing of the physiological data was performed with the IOX software (EMKA) and expressed in grams (g). The data are positive for the contractions and negative for the relaxations. The pEC50 values were calculated as the negative log of the half-maximum effective concentration (EC50 values). The second concentration–response curve induced by NE was expressed as % of the Emax of the initial curve. All data are presented as means ± SEM derived from (n) patients. The concentration–response curves obtained were analysed by two-way ANOVA and Student Newman–Keuls post hoc test using within-patient comparison. The pEC50 and Emax were analysed by paired Student’s t-test. If there was more than one preparation (same condition and protocol) for one patient, the results were averaged before statistical analysis. A P-value <0.05 was considered statistically significant.

2.8 Compounds
IL-1β was obtained from PromoCell. ACh, NE, LPS, antibiotics, and antimycotic were purchased from Sigma-Aldrich. Etoricoxib was obtained from Sequoia Research Products Ltd. U46619 [GenBank] , SC-560, and DuP-697 were obtained from Cayman, DFU was a gift from Merck Frosst.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
3.1 Effect of interleukin-1β and lipopolysaccharide on the vascular reactivity
The first concentration–response curve and the maximal contraction induced by NE in human IMA were significantly reduced after 6 (n = 9) and 24 h (n = 20) of culture with IL-1β and LPS while the pEC50 was not affected (Figure 1A; Table 1). No difference in curves obtained with U46619 [GenBank] , ACh and KCl was observed between preparations cultured during either Control or inflammatory conditions (Figure 1B–D; Table 1; n = 4–7). In addition, independent of the treatment, the IMA preparations were significantly less sensitive to NE at 24 h in comparison with fresh preparations (0 h; Table 1).


Figure 1
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  		Figure 1 Reactivity of human internal mammary artery preparations induced by norepinephrine (A, n = 20), U46619 [GenBank] (B, n = 5), acetylcholine (C, n = 7), and KCl (D, n = 4) (first concentration–response curve) after 24 h of culture under non-inflammatory (Control) or inflammatory conditions [IL-1β (interleukin-1β) + LPS (lipopolysaccharide)]. (*) Indicates P < 0.05, ANOVA. See Table 1 for pEC50 and Emax values.

 


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  		Table 1 Effect of interleukin-1β and lipopolysaccharide on human mammary artery reactivity

 
3.2 Effects of cyclooxygenase inhibitors on norepinephrine-induced vasoconstrictions
To evaluate the involvement of each COX activity in the control of the vascular tone, the second concentration–response curve to NE was obtained with or without COX-1 and COX-2-selective inhibitors. The COX-2-selective inhibitors (DFU, DuP-697 and Etoricoxib) increased the contractions induced by NE only in IMA preparations treated with IL-1β and LPS for 6 or 24 h (Figure 2; Table 2; n = 5–8). No difference in pEC50 was observed between Control and inflammatory conditions. The selective COX-1 inhibitor (SC-560) increased significantly the contraction induced by NE either after 6 or 24 h of culture (Table 2; n = 5). However, in presence of SC-560, the NE concentration–response curves were not different between preparations cultured in Control and inflammatory conditions after 6 or 24 h of culture (Figure 2; Table 2; n = 5).


Figure 2
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  		Figure 2 Effect of selective cyclooxygenase inhibitors on human internal mammary artery contraction induced by norepinephrine (second concentration–response curve) after 24 h of culture under non-inflammatory (Control) or inflammatory conditions [IL-1β (interleukin-1β) + LPS (lipopolysaccharide)]. (*) Indicates P < 0.05, ANOVA, n = 5. The Emax was expressed as % of the maximal norepinephrine contraction obtained in the first concentration–response curve. Similar results have been obtained after 6 h of culture. See Table 2 for pEC50 and Emax values.

 


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  		Table 2 Effect of cyclooxygenase inhibitors on human mammary artery vasoconstrictions induced by norepinephrine

 
3.3 Norepinephrine-induced vasoconstriction in absence of endothelium
To evaluate the role of the endothelium in the contraction induced by NE, concentration–response curves to NE were obtained without endothelium before and after 30 min incubation with Etoricoxib (1 µmol/L; Figure 3; Table 3). In absence of endothelium, the maximal contraction induced by NE in human IMA was significantly reduced after 24 h of culture with IL-1β and LPS (Figure 3A; Table 3; n = 7). In these preparations, the COX-2-selective inhibitor (Etoricoxib) increased significantly the contractions induced by NE only in IMA preparations treated with IL-1β and LPS for 24 h (Table 3, Figure 3B, n = 7). No difference in the sensitivity (pEC50) of these preparations to NE was observed between Control and inflammatory conditions.


Figure 3
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  		Figure 3 Contraction induced by norepinephrine of human internal mammary artery where the endothelium has been rubbed after 24 h culture under non-inflammatory (Control) or inflammatory conditions [IL-1β (interleukin-1β) + LPS (lipopolysaccharide)]. The panel (A) shows the first norepinephrine concentration–response curve, n = 7. The panel (B) shows the second norepinephrine concentration–response curve after 30 min incubation with Etoricoxib (1 µmol/L) a selective COX-2 inhibitor. (*) Indicates P < 0.05, ANOVA, n = 7. See Table 3 for pEC50 and Emax values.

 


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  		Table 3 Contraction induced by norepinephrine in human internal mammary arterial preparations without endothelium

 
3.4 Effect of interleukin-1β and lipopolysaccharide on prostaglandin E2, 2,3-dinor-6-keto-prostaglandin F1{alpha} and thromboxane B2 release
IL-1β and LPS caused a significant increase in both PGE2 and 2,3-dinor-6-keto-PGF1{alpha} but not TxB2 released by IMA in culture medium after 6 or 24 h (Figure 4A, n = 4–5). When compared with the Control preparations, the PGE2 production was 2.1-fold greater after 6 h of culture and 3.3-fold greater after 24 h of culture. While the 2,3-dinor-6-keto-PGF1{alpha} production was 1.9- and 2.3-fold greater after 6 and 24 h of cultures, respectively.


Figure 4
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  		Figure 4 Measurement of PG (prostaglandin) E2, 2,3-dinor-6-keto PGF1{alpha} and Tx (thromboxane) B2 released by human internal mammary arteries into the media in culture (A) and in organ bath (B) experiments under non-inflammatory (Control) and inflammatory conditions [IL-1β (interleukin-1β) + LPS (lipopolysaccharide)]. DuP-697 (0.5 µmol/L), a selective COX-2 (cyclooxygenase-2) inhibitor, decreased the prostaglandin release in organ bath medium. The results derived from n = 4–5 individual; (*) indicates data significantly different vs. their respective Control (P < 0.05, Student’s paired t-test).

 
The PGE2 and 2,3-dinor-6-keto-PGF1{alpha} released was also significantly increased in organ bath medium containing preparations cultured for 6 and 24 h in inflammatory conditions (Figure 4B, n = 4). When compared with the Control preparation, the PGE2 production was 3.8-fold greater after 6 h and 13-fold greater after 24 h of culture. While the 2,3-dinor-6-keto-PGF1{alpha} production was 4- and 3.9-fold greater after 6 and 24 h of culture, respectively.

3.5 Effect of interleukin-1β and lipopolysaccharide on cyclooxygenase-1 and cyclooxygenase-2 expression
The western blot analysis (Figure 5) shows that the COX-1 isoform was detected similarly at either 6 or 24 h in the IMA preparations incubated in absence or presence of IL-1β and LPS. In contrast, COX-2 protein was slightly expressed in the IMA after 6 h of culture in both conditions. However, after 24 h, the expression of COX-2 was markedly induced in tissues treated with IL-1β and LPS and mildly expressed in Control condition. Similarly, data derived from the immunohistochemical experiments (Figure 6) demonstrate that the COX-1 isoform was present throughout the entire vascular wall in either Control tissues or preparations exposed to the inflammatory conditions. There was no difference between 6 and 24 h of culture (Figure 6A and B). The COX-2 isoform was markedly expressed in presence of IL-1β and LPS and was mainly localized in the endothelium as well as in the smooth muscle layers after 6 h and in the entire vascular wall after 24 h of culture. In contrast to COX-1, the COX-2 isoform was not expressed in fresh IMA preparations using immunohistochemistry experiments (0 h, n = 4, data not shown).


Figure 5
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  		Figure 5 Effect of IL-1β (interleukin-1β) + LPS (lipopolysaccharide) on COX-1 (cyclooxygenase-1) and COX-2 (cyclooxygenase-2) expression in human internal mammary arteries. The western blot analysis shows an induction of COX-2 isoform under inflammatory conditions (IL-1β + LPS) after 24 h of culture compared with non-inflammatory conditions (Control). COX-1 and COX-2 were detected using monoclonal antibodies. Purified ovine COX-1 or COX-2 was used as standard. A representative experiment from n = 3 is presented.

 


Figure 6
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  		Figure 6 Immunostaining of COX-1 (cyclooxygenase-1) and COX-2 (cyclooxygenase-2) in human internal mammary artery after 6 h (A, n = 4) and 24 h (B, n = 4) of culture under non-inflammatory (Control) and inflammatory conditions [IL-1β (interleukin-1β) + LPS (lipopolysaccharide)]. Positive immunoreactions are observed as a brown precipitate. a, adventitia; m, media; i, intima; l, lumen (magnification x20). Insets: high-power views show a positive staining on endothelium (black arrows).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
The present work demonstrates that the induction of COX-2 in response to acute inflammatory stimuli caused a decrease in NE reactivity in human IMA. This reduction was accompanied by an increase in PGE2 and PGI2 release. Under this inflammatory condition, the human IMA tone induced by NE was restored and increased by selective COX-2 inhibitors associated with a decrease in PGE2 and PGI2 synthesis. These observations suggest that the COX-2 inhibition acts specifically at the site of the vascular smooth muscle to reduce the release of PGE2 and PGI2 resulting in an increased vascular responsiveness to NE. Therefore, a considerable part of the inflammatory detrimental effects of COX-2 inhibitors in cardiovascular disease may be due, in part, to their direct actions at the level of the vascular smooth muscle. This activity appears to be independent of a platelet TxA2 release mechanism.

Numerous studies have shown that both COX isoforms are expressed by the majority of the human blood cells and by the vascular wall.7,8,10,23 In human arteries, the COX isoforms are present in endothelial18,24–26 and vascular smooth muscle cells.9,19,23 In most of these studies, the COX-2 expression was induced by inflammatory stimuli such as IL-1β and LPS and was time dependent. The expression and localization of COX-1 and COX-2 have been examined in human IMA after 6 and 24 h of culture. We have detected the presence of COX-1 isoform using immunohistochemistry in the endothelial and smooth muscle cells under Control and inflammatory conditions as shown in the human aorta and carotid.7,23 Only under inflammatory conditions, a marked COX-2 immunostaining in IMA was observed which was localized both in the endothelial and smooth muscle layers after 6 h and in the entire of vascular wall after 24 h of culture. This observation suggests that endothelial and smooth muscle cells are more sensitive to express COX-2 under inflammatory conditions when compared with adventitia. Again, these results are in accordance with previous publications using pathological vessels (atherosclerosis or aneurysm) where these inflammatory conditions have induced COX-2 expression.7,10,12,23 In these studies and the present report, immunohistochemical experiments have revealed that COX-2 was mainly localized in the intimal and the medial layers. In addition, our western blot experiments have shown a COX-2 induction after 24 h of culture, however, after 6 h of culture, the COX-2 isoform was slightly detected. On human vascular isolated cells, several reports have demonstrated by western blot analysis an induction of COX-2 isoform only 2 h after inflammatory stimulation.9,18,26 The absence of induction in our study at 6 h using western blot analysis may be explained by a lower or a delayed efficiency of IL-1β and LPS on whole tissue in comparison with isolated cells. Furthermore, the discrepancy in COX-2 detection observed at 6 h between western blot analysis and immunohistochemistry (Figures 5 and 6) could be attributed to a difference in sensitivity of the techniques used.

In several studies, the induction of COX-2 is accompanied by a preferential increase in PGI2 and PGE2 synthesis but not TxA2.6,9,26 In the present report, we have also measured increased productions of PGI2 and PGE2 metabolites in our media under inflammatory conditions while TxA2 level was unchanged. In addition, this increase in PG production is clearly related to COX-2 activity since DuP-697 reduced significantly the PG release. The production was higher in organ bath than in culture medium, this difference may be due to the methodology used, oxygen density, run-time of release and/or stretch tone of IMA.

The role of COX-2 activity in the control of vascular tone was the focus of our study and may be related to the effects on hypertension and cardiovascular disease. In the present report, we have shown that the inflammatory conditions reduce significantly the IMA reactivity induced by NE while other stimuli such as U46619 [GenBank] , ACh and KCl remained unaffected. After 6 and/or 24 h a potent induction of COX-2 activity was detected in our preparations. Together, these results suggest an involvement of COX-2 products in the regulation of NE contraction. In fact, a greater production of PGE2 and PGI2 may be associated to the NE stimulation under inflammatory conditions. These PGs will increase vasorelaxation and consequently will reduce the contractile effect induced by NE via the {alpha} receptors. We demonstrated also that the reduction of NE contraction under inflammatory conditions is endothelium independent and mainly related to COX-2 induction in the smooth muscle layer. Another work has also shown no difference in the IMA reactivity to NE after endothelium removing.27 The sensitivity of IMA to NE was decreased (~10-fold) after 24 h of culture in non-inflammatory condition when compared with 0 and 6 h of culture. This effect may be due to a low COX-2 induction as shown in Figure 5 in Control after 24 h despite the presence of antibiotics and the absence of IL-1β and LPS. The decrease in the vascular tone observed under inflammatory conditions in the present report is in accordance with other studies on animals model where the vascular reactivity was reduced under inflammatory conditions.22,28 However, these reductions appear to be variable depending on the agonist, the vessel or the species studied. The contraction of isolated porcine coronary artery induced by U46619 [GenBank] and KCl was reduced (20–30%) following overnight culture with 100 µg/mL LPS.22 Another work from Virdis et al.28 has demonstrated that the contraction of isolated mesenteric artery induced by NE was also reduced in LPS-treated rats (6 h, in vivo). In this model, the relaxant response induced by ACh was also reduced, an effect restored after the treatment with DFU.

The present data demonstrate that the reduced NE vasoconstriction is due to COX-2 activity since the three selective COX-2 inhibitors (DFU, DuP-697, and Etoricoxib) restored and increased this contraction, an effect endothelium independent. The selectivity of these inhibitors, at the concentrations employed in our experiments, has been previously described since DFU, DuP-697, and Etoricoxib were potent inhibitors of the production of PGE2 in Chinese hamster ovary cells expressing human COX-2 but not in COX-1 cells.29,30 Several in vivo studies have shown an effect of COX-2 inhibition on blood pressure. For example, chronic injection of rofecoxib causes an increase in blood pressure in spontaneously hypertensive rats, an effect dependant on the inhibition of COX-2 and prostacyclin synthesis.31 In addition, a recent meta-analysis of ~45 000 patients in 19 clinical trials show that selective COX-2 inhibitors significantly elevate systolic blood pressure (+3.85 mmHg) and a similar but reduced effect was observed with the non-selective COX inhibitors.32 Finally, injection of LPS in Sprague–Dawley rats caused a marked decrease in systolic arterial pressure, from 128 to 79 mmHg.33 In this latter study, the decreased systemic arterial pressure induced by LPS was restored in rats receiving rofecoxib, this effect was accompanied by a decrease in PGE2 and 2,3-dinor-6-keto-PGF1{alpha} levels in plasma. Since essential hypertension is associated with an increase in the vascular tone, the previous results with COX-2 inhibitors and the increased blood pressure in rats are supported by the observations in human vascular preparations in the present report.

In contrast to the effect of COX-2 inhibitors between Control and inflammatory conditions, the NE contraction was not different after treatment with a selective COX-1 inhibitor (SC-560). These observations are in accordance with a previous report where mesenteric artery contractions in presence of SC-560 were not different between normal and LPS-treated rats.28 Our results on the vascular tone are consistent with those obtained by immunohistochemistry and western blot analysis where the COX-1 isoform was unchanged under both conditions. Whatever are the culture conditions, the COX-1 inhibitor increased significantly the NE contraction (Table 2). This effect may be explained by a decrease in a vasorelaxant PG produced by COX-1 such as PGI2. In fact, previous works have demonstrated that NE stimulation increases the PGI2 release in healthy human vessels.34,35

The PGs, i.e. PGI2 and PGE2, produced by COX-2 are responsible for vasodilatations and reduce the vascular reactivity to NE. The induction of PGE Synthase (PGES) may be associated to an increase in PGE2 concentration under inflammatory conditions. An inducible isoform named mPGES-1 has been detected in human arterial smooth muscle cells after 24 h of culture with IL-1β.36 This induction was also accompanied with an increase in PGE2 release. In these conditions, PGI2 synthesis was mainly dependent on COX-2 activity, as PGIS expression was not modified by IL-1β.18,36 However, an increased expression of relaxant PG receptors (IP, DP, EP2, and EP4) may provide an alternative explanation. For example, an induction of the EP2 and/or EP4 receptor expression associated with the increase in the PGE2 production would reduce the vasoconstriction induced by NE. The overexpression of EP2 and/or EP4 receptor mRNA has been detected in human cervical and synovial fibroblasts after the treatment with IL-1β.37 Another study in the vascular wall has shown that the overexpression of EP4 receptor was associated with enhanced inflammatory reaction in symptomatic atherosclerotic plaque in human carotid artery in comparison with asymptomatic plaque.38

In conclusion, we demonstrate that the induction of COX-2, associated with the increase in PGI2 and PGE2 synthesis, reduces the vascular tone in human IMA to NE. This reduction was reversed and the vascular tone increased after treatment with selective COX-2 inhibitors. These results suggest that the cardiovascular risk of coxibs which emerged from clinical trials may be, in part, associated with a direct increase in the vascular tone which is independent of any TxA2 release from platelets.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
This work was supported by Institut National de la Santé et de la Recherche Médicale (INSERM), France.


   Acknowledgements
 
The authors would like to thank Dr Richard Friesen for providing DFU, a Merck Frosst compound.

Conflict of interest: none declared.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 

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