Skip Navigation

Cardiovascular Research 2003 57(1):109-118; doi:10.1016/S0008-6363(02)00657-0
© 2003 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Molero, L.
Right arrow Articles by López-Farré, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Molero, L.
Right arrow Articles by López-Farré, A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2003, European Society of Cardiology

Endothelin-1 induced proinflammatory markers in the myocardium and leukocytes of guinea-pigs

Role of glycoprotein IIB/IIIA receptors

Laura Molero, Jeronimo Farré, Antonio García-Mendez, Petra Jiménez Mateos-Caceres, Carolina Carrasco Martín, Inmaculada Millás, Felipe Navarro, Manuel Córdoba, Santos Casado and Antonio López-Farré*

Cardiovascular Research Laboratory, Fundación Jiménez Díaz, Av. Reyes Católicos, 2, Madrid, 28040 Spain

* Corresponding author. Tel.: +34-91-550-4821 alopeza{at}fjd.es

Received 18 April 2002; accepted 3 September 2002


   Abstract
Top
 Abstract
1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Aim:To assess whether endothelin-1 (ET-1) induces the in vivo expression of inflammatory-related proteins, namely cyclooxygenase-2 (COX-2) and tissue factor, in the myocardium and circulating leukocytes of guinea-pigs. The involvement of platelets was also analyzed. Methods: ET-1 (0.013 µg/min) was infused to male guinea-pigs for 45 min in the presence and absence of tirofiban, a nonpeptidic blocker of the glycoprotein IIb/IIIa receptor (GPIIb/IIIa). Tissue factor and COX-2 expression were determined by Western blot. Results: No changes in mean arterial pressure and heart rate were detected. ET-1-infused guinea-pigs showed a marked increase in the number of platelets expressing activated GPIIb/IIIa receptors (0.8±0.03% vs. 6.5±0.2%; P<0.05). Tirofiban (10 µg/Kg bw/min) blunted ex vivo platelet aggregation in response to ADP, although only partially reduced COX-2 and tissue factor expression in both the myocardium and leukocytes of ET-1-infused guinea-pigs. The myocardium of platelet-depleted guinea-pigs also showed a reduced COX-2 expression after ET-1 infusion (57±3% reduction; P<0.05). In vitro studies demonstrated that platelets (107 and 109 platelets/well) enhanced ET-1 (10–7 mol/l)-induced COX-2 expression in heart slices. Conclusion: ET-1 stimulated in vivo the expression of the pro-inflammatory proteins COX-2 and tissue factor in the myocardium and in leukocytes by a mechanism GPIIb/IIIa platelet receptors.

KEYWORDS Endothelial factors; Endothelins; Infection/inflammation; Leukocytes; Myocytes; Platelets


   1 Introduction
Top
 Abstract
 1 Introduction
2 Material and methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Endothelial dysfunction and atherosclerotic plaque rupture initiate platelet activation and result in a conformational change of glycoprotein IIb/IIIa (GPIIb/IIIa) receptors on the platelet surface [1,2]. The GPIIb/IIIa receptor binds circulating fibrinogen, von Willebrand factor and other adhesive proteins leading to platelet aggregation and thrombosis [1,3]. Recent clinical data have suggested that GPIIb/IIIa receptor blockade reduces the incidence of cardiac events in patients with unstable angina or non-Q-wave myocardial infarction [4,5]. Platelet activation is tightly regulated by products released from the endothelium, including endothelin-1 (ET-1) [6,7].

ET-1 is a potent constrictor of vascular smooth muscle cells [8]. We and others have demonstrated that ET-1 also stimulates in vitro adherence of leukocytes to endothelium, inducing the expression of adhesion molecules on endothelial cells and leukocytes, and triggers the release of interleukin-6, suggesting a pro-inflammatory action [9–11].

Endothelial production of ET-1 is increased in the presence of coronary risk factors such as atherosclerosis, hypertension and hypercholesterolemia and also during myocardial ischemia [12–14], when platelet activation and myocardial inflammation seem to play a main role. However, to date, little information is available on the in vivo proinflammatory action of ET-1. In the present study, we examined whether ET-1 induces in vivo expression of inflammatory proteins, namely cyclooxigenase-2 (COX-2) and tissue factor [15,16], in the myocardium and circulating leukocytes from guinea-pigs. The involvement of platelets was also analyzed.


   2 Material and methods
Top
 Abstract
 1 Introduction
 2 Material and methods
3 Results
 4 Discussion
 Acknowledgments
 References
 
2.1 In vivo experiments
Male Dunkin–Hartley guinea-pigs (average weights 400±30 g) were used in the study approved by the Animal Research Commettee. All surgical procedures were performed after intraperitoneal injection of sodium pentobarbital (37.5 mg/Kg bw i.p.). Catheters were inserted into the jugular vein and carotid artery. Arterial pressure was monitored continuously by a pressure transducer (Power Laboratory System, Cybertec). A 22-gauge needle connected to an infusion pump was inserted through the carotid artery to the aorta ({approx}2 cm below the carotid bifurcation). Continuous isotonic saline (0.1 ml/min) was administered during a 30-min equilibration period. Afterwards, guinea-pigs were divided into three groups: (a) ten guinea-pigs were infused with isotonic saline only (control group); (b) ten guinea-pigs were infused with ET-1 (0.013 µg/min) for 45 min in isotonic saline; we have previously demonstrated that this does does not modify arterial pressure [9]; (c) ten guinea-pigs were infused with ET-1 (0.013 µg/min) for 45 min with a non-peptidic blocker of GPIIb/IIIa receptors, tirofiban (10 µg/Kg bw/min). Saline and tirofiban were continuously infused for 3 h after ET-1 infusion. Previous works have shown that continuous infusion of tirofiban for 1–6 h prevents platelet activation [17,18]. After this second infusion, the catheters were removed and the animals allowed to recover.

Twenty four hours later guinea-pigs were anesthetized and exsanguinated. Blood was collected into EDTA-tubes and the left ventricle was removed and quickly frozen in liquid nitrogen for molecular biology determinations.

2.2 Leukocyte isolation
Blood samples were obtained by aortic puncture. Circulating leukocytes were isolated by Ficoll/Hypaque centrifugation and quickly frozen at –80 °C for molecular biology determinations. Leukocyte isolation and manipulation was always performed under sterile conditions.

2.3 Determination of COX-2 and tissue factor expression
COX-2 and tissue factor expression was analyzed by Western blot. In brief, the frozen myocardium and leukocytes were solubilized in Laemmli buffer [19] containing 2-mercaptoethanol. Proteins (20 µg/lane) were separated in denaturing SDS 10% polyacrylamide gels. Western blot analysis was performed with a mouse monoclonal antibody against COX-2 (1:1000) (Alexis Biochemicals) and a murine tissue factor monoclonal IgG1 antibody (American Diagnostic), as reported [20,21]. Specific COX-2 and tissue factor proteins were detected by enhanced chemiluminescence (ECL, Amersham International) and evaluated by densitometry (Molecular Dynamics). A parallel gel with identical samples was run and, after blotting onto nitrocellulose, Western blot analysis was performed with a β-actin monoclonal antibody (1:5000) (Sigma–Alchich). Prestained protein markers (Sigma) were used for molecular mass determinations.

2.4 COX-2 and tissue factor activity in ex vivo incubated leukocytes
Leukocytes isolated from the three groups of guinea-pigs (24 h after the infusions) were incubated ex vivo in RPMI medium supplemented with 0.25% bovine serum albumin for 3 h at 37 °C. The supernatants were recovered and centrifuged (2500 rpm, 10 min) to determine 6-keto PGF1{alpha} (the stable metabolite of prostacyclin) and prostaglandin E2, as index of COX-2 activity, using radioimmunoassay (RIA) systems (Amersham Life Science, UK) and tissue factor using enzyme-linked immunosorbent assay (ELISA) (Movaco SA, Spain).

2.5 Determination of activated GPIIb/IIIa
Activated GPIIb/IIIa (PAC-1) expression on platelets was quantified in whole blood obtained from control and ET-1 (0.013 µg/min)-infused guinea-pigs by flow cytometry. In brief, 3 ml of EDTA anticoagulated whole blood (150 µl) were immediately fixed with 1% paraformaldehyde. To detect platelets with activated GPIIb/IIIa receptors, samples were incubated with an FITC-labeled monoclonal antibody (IgM) PAC-1 (1 µg, Becton Dickinson, San Jose, CA, USA) for 30 min at room temperature. The samples were analyzed in a FACS flow cytometer (Becton Dickinson) as described [9]. A minimum of 5000 cells for each sample were analyzed. Mean activated GPIIb/IIIa positive fluorescence for the entire platelet population was expressed in arbitrary fluorescence units. The percentage of cells positive for activated GPIIb/IIIa was generated by substracting an isotype control (mouse FITC-labeled Ig M).

2.6 Ex vivo platelet aggregation
To determine the dose of tirofiban that prevented platelet activation in guinea-pigs, we evaluated ex vivo the ability of ADP to aggregate platelets from the three groups of guinea-pigs that previously were infused with different doses of tirofiban (1, 3 and 10 µg/Kg bw/min) for 3 h. Immediately after completing the GPIIb/IIIa blocker infusion, whole blood was collected in 10% acid–citrate–dextrose and platelet-rich plasma (PRP) was obtained as already described [22]. Platelet activation by ADP (10–6 mol/l) was measured by an aggregometer (Chronolog, 4 channels) as described elsewhere [6,22]. Only the value obtained by turbidimetry at 3 min was used for the calculations.

2.7 Immunohistochemical studies
Sections of the left ventricle of three guinea-pigs from each experimental group were perfused with 50 ml fixative solution containing 4% paraformaldehyde in serum saline (1:1 vol/vol). The aortas were embedded in paraffin wax and sectioned as previously reported [23,24]. COX-2 protein was identified by a polyclonal antibody against COX-2 (Dako Corporation, Carpinteria CA) at the dilution of 1:100 and subsequent reactions with biotinylated antiserum to rabbit IgG (Dako Corporation) as reported [23,24].

2.8 Guinea-pig antiplatelet-enriched serum production
Antiplatelet-enriched serum was obtained as described [23]. In brief, pure isolated platelets obtained from control guinea-pigs were homogenized in Freund's complete adjuvant and injected subcutaneously in rabbits. Rabbit plasma was collected by plasmapheresis 10 days after a second immunization. Nonimmune serum was obtained from nonimmunized rabbits and served as control (control IgG).

Guinea-pigs were injected with the antiplatelet (Apab)-enriched serum or control serum i.p. (30 mg/100 g bw) 24 h before ET-1 infusion. This dose of Apab results in severe thrombocytopenia with platelet counts <10.000/mm3 within 24 h, representing an 85% reduction (P<0.01) relative to platelet counts in control IgG serum-treated guinea-pigs.

2.9 In vitro coincubation of PRP with heart slices
Heart slices from the heart of control guinea-pigs were preincubated in RPMI medium supplemented with 2.5% fetal calf serum. The slices were then incubated at 37 °C with 10–7 mol/l ET-1 in the presence and in the absence of PRP (107 and 109 platelets/well) for 1 h at 37 °C in a cocultured system. The cocultured system was prepared by placing transwell inserts containing the platelets into wells containing the heart slices. In this coculture system, the medium was shared by both types of cells and made possible further processing of the heart slices alone. After 1 h of coincubation, the transwell containing platelets was removed and heart slices were incubated at 37 °C for 23 h and recovered and frozen quickly in liquid nitrogen for COX-2 protein determination.

To test the effect of inactivated and ET-1-activated platelets on COX-2 expression, we studied the time in which isolated platelets remained inactive once PRP was isolated from whole blood. For this purpose, PRP was labeled with [3H]-serotonine (0.5 µCi/ml) as reported [25]. After extensive washing, platelets were resuspended in platelet-poor plasma and incubated at 37 °C. At different times, a sample of the platelet-incubated medium was centrifuged at 2500 rpm for 10 min and the supernatant measured in a scintillition counter. One hour after incubation at 37 °C, the percentage of [3H]-serotonine released from platelets was 5.0±0.08%, while 3 h after the percentage of [3H]-serotonine released from platelets was 41.4±6.4% (P<0.05). Therefore, the platelets were coincubated with heart slices for only 1 h.

2.10 Electrophoretic mobility-shift assay (EMSA)
Nuclear protein extracts were obtained from heart slices that previously had been incubated in vitro with 10–7 mol/l ET-1 for 1 h at 37 °C in the presence and absence of platelets (109 platelets/well) and tirofiban (5x10–7 mol/l). Nuclear protein extracts were isolated as reported [26,27] and mixed with an oligonucleotide corresponding to a double-stranded SP-1 consensus oligonucleotide (ATT CGA TCG GGG CGG GGC GAG C-3' Promega). The SP-1 oligonucleotide was end-labelled using [{gamma}-32P]-ATP and T4 polinucleotide kinase. Standard binding reactions were performed by incubating 10 µg of nuclear extracts in 20 µl of 10 mmol/l Tris–HCl, pH 7.5, containing 10 mmol/l NaCl, 1 mmol/l dithiothreitol, 1 mmol/l EDTA, 10 mmol/l MgCl2, 20% (v/v) glycerol and 50.000 dpm of 32P-labeled SP-1 oligonucleotide (approximately 1 pmol) for 20 min at room temperature. Nuclear extracts from HELA were used as positive control. After incubation, the samples were loaded onto 4% polyacrilamide gel and run at a constant current of 100 v in 0.5xTris-borate-EDTA (TBE) at 4 °C. Gels were dried and placed on film at –70 °C.

2.11 Statistical methods
Results are represented as mean±S.E.M. Unless otherwise stated, each value corresponds to samples obtained from a minimum of eight different animals. To determine the statistical significance of our results, we performed an ANOVA with Bonferroni's correction for multiple comparisons or a Student's t-test (paired or unpaired). A P<0.05 was considered statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
4 Discussion
 Acknowledgments
 References
 
3.1 COX-2 and tissue factor expression after ET-1 infusion
No changes in mean arterial pressure (control: 71±3; ET-1: 77±4 mmHg, pNS), heart rate (control: 192±5; ET-1: 202±7 bpm, pNS) and hematocrit (control: 45±2%; ET-1: 44±1%, pNS) were observed after the administration of ET-1.

ET-1 infusion stimulated COX-2 expression in guinea-pigs myocardium (Fig. 1). A marked COX-2 expression was obtained 24 h after ET-1 infusion and remained elevated 48 and 72 h after ET-1 infusion (Fig. 1). Therefore, the value obtained 24 h after ET-1 infusion was used for most of the following experiments. The monoclonal COX-2 antibody used in our experiments did not cross-react with the COX-1 isoform because a COX-2 band was undetectable in a homogenate of guinea-pig platelets (Fig. 2a). Tissue factor expression was also found increased in the myocardium of ET-1-infused guinea-pigs (Fig. 2a and b). No changes in β-actin protein expression were observed in the left ventricle of the different groups of animals (Fig. 2a).


Figure 1
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Representative Western blot demonstrating the expression of cyclooxygenase-2 (COX-2) in the left ventricle of guinea-pigs. Guinea-pigs were infused with endothelin-1 (ET-1, 0.013 µg/min) for 45 min. At 24, 48 and 72 h after infusion, the myocardium was removed. The bottom shows the bar graph representing the densitometric analysis of the Western blots. Results are represented as mean±S.E.M. of eight different guinea-pigs for each experimental group. * P<0.05 with respect to control.

 

Figure 2
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 (A) Representative Western blot demonstrating the expression of cyclooxygenase-2 (COX-2), tissue factor and β-actin in the left ventricle of guinea-pigs. Guinea-pigs were infused with ET-1 (0.013 µg/min) for 45 min in the presence and absence of the GPIIb/IIIa blocker, tirofiban (10 µg/Kg bw/min for 3 h). Twenty four hours after infusion, the myocardium was removed. Platelets (PLT) were used as negative control of COX-2 expression. (B) Bar graph showing the densitometric analysis of the Western blots. Results are represented as mean±S.E.M. of eight different guinea-pigs for each experimental group. * P<0.05 with respect to controls. * P<0.05 with respect to ET-1-infused guinea-pigs.

 
The Western blot results were confirmed in immunohistochemical studies. COX-2 protein expression was greater in the myocardium of ET-1-infused guinea-pigs than in control animals (Fig. 3a and b). Control inmmunostaining in which the primary antibody was replaced with normal rabbit serum produced no positive signal. Immunolocalization of COX-2 demonstrated that cells expressing COX-2 were also positively stained with sarcomeric {alpha}-actin indicating they were cardiomyocytes (data not shown). Furthermore, Giemsa and hematoxylin and eosin staining of the myocardium showed absence of infiltrated leukocytes in ET-1-infused guinea-pigs.


Figure 3
View larger version (109K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Representative photomicrographs of cross-sections of the left ventricle of (A) Control (B) ET-1 (0.013 µg/min)-infused and (C) tirofiban (10 µg/Kg bw/min)-treated ET-1-infused guinea-pigs. The expression of COX-2 was detected in paraffined sections obtained 24 h after infusion. COX-2 is indicated by arrows. Bar=25 µm.

 
ET-1 infusion also increased tissue factor and COX-2 expression in circulating leukocytes (Fig. 4a and b).


Figure 4
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 (A) Representative Western blot demonstrating that expression of cyclooxygenase-2 (COX-2) and tissue factor in circulating leukocytes of guinea-pigs. Guinea-pigs were infused with ET-1 (0.013 µg/min) for 45 min in the presence and absence of the GPIIb/IIIa blocker, tirofiban (10 µg/Kg bw/min for 3 h). Twenty four hours after infusion, leukocytes were obtained for molecular determinations. (B) Bar graph showing the densitometric analysis of the Western blot. Results are represented as mean±S.E.M. of eight different guinea-pigs for each experimental group. * P<0.05 with respect to controls. * P<0.05 with respect to ET-1-infused guinea-pigs.

 
3.2 GPIIb/IIIa inhibition and COX-2 and tissue factor expression
Activated GPIIb/IIIa receptors were expressed in less than 0.8±0.03% of platelets from control guinea-pigs. The number of platelets expressing activated GPIIb/IIIa was significantly increased after 45 min of ET-1 (0.013 µg/min) infusion (6.5±0.2%, P<0.05).

Afterwards, we examined the involvement of platelets and GPIIb/IIIa receptors in ET-1-dependent inflammatory response, blocking GPIIb/IIIa receptors with tirofiban.

The tirofiban dose used to inhibit platelet activation was chosen based on previous ex vivo aggregometry experiments with platelets from guinea-pigs treated with increasing tirofiban doses. A marked reduction of ADP-induced platelet aggregation was obtained with 3 µg/Kg bw/min tirofiban. Ten µg/Kg bw/min tirofiban blunted ADP-induced platelet aggregation (Table 1) and was used in the following studies.


View this table:
[in this window]
[in a new window]

  		Table 1 Effect of in vivo administration of tirofiban on ex vivo ADP-induced guinea-pig platelet aggregation

 
GPIIb/IIIa receptor blockade with tirofiban (10 µg/Kg bw/min) significantly reduced both COX-2 and tissue factor expression in the myocardium of ET-1-infused guinea-pigs (Fig. 2a and b). However, COX-2 and tissue factor expression in the myocardium remained elevated with respect to control animals (Fig. 2a and b). Increased concentrations of tirofiban (20 and 40 µg/Kg bw/min) demonstrated no greater inhibition of COX-2 or tissue factor expression than 10 µg/Kg bw/min (data not shown).

Immunohistochemical analysis also showed a reduction of COX-2 expression in the myocardium of tirofiban-treated ET-1-infused guinea-pigs, although it remained elevated with respect to control animals (Fig. 3a, b and c). No expression of GPIIb/IIIa receptors was found in the myocardium of ET-1-infused guinea-pigs by both Western blot and immunohistochemical analysis.

Leukocytes obtained from tirofiban (10 µg/Kg bw/min)-treated ET-1-infused guinea-pigs also showed a significant reduction of COX-2 and tissue factor expression (Fig. 4a and b). However, expression of these proteins remained elevated with respect to leukocytes from control guinea-pigs (Fig. 4a and b).

After in vivo infusion of ET-1, ex vivo incubated leukocytes released significantly larger amounts of 6-ketoPGF1{alpha}, prostaglandin E2 and tissue factor than leukocytes from control animals (Table 2). Tirofiban administration partially reduced 6-keto-PGF1{alpha}, prostaglandin E2 and tissue factor production by leukocytes from ET-1-infused guinea-pigs (Table 2).


View this table:
[in this window]
[in a new window]

  		Table 2 Prostacyclin, prostaglandin E2 and tissue factor released from leukocytes

 
3.3 Platelets and ET-1-induced inflammatory response
Apab-treated guinea-pig myocardium showed a reduced COX-2 expression after ET-1 infusion when compared with control IgG-serum-treated ET-1-infused guinea-pigs (Fig. 5a and b).


Figure 5
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 (A) Representative Western blot demonstrating COX-2 expression in the left ventricle of guinea-pigs. Guinea-pigs were injected with antiplatelet IgG-enriched (Apab) serum or control IgG 24 h before ET-1 (0.013 µg/min) infusion. (B) Bar graph showing the densitometric analysis of the Western blot. Results are represented as mean±S.E.M. of eight different guinea-pigs for each experimental group. * P<0.05 with respect to controls. * P<0.05 with respect to ET-1-infused guinea-pigs.

 
In in vitro incubated heart slices, 107 and 109 platelets alone failed to modify COX-2 expression (Fig. 6a). ET-1 (10–7 mol/l) stimulated COX-2 expression in in vitro incubated myocardium (Fig. 6b) which was significantly enhanced by platelets in a concentration-dependent manner (Fig. 6b).


Figure 6
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Representative Western blot demonstrating the effect of platelets (PLT) on the COX-2 expression in in vitro incubated left ventricle slices from guinea-pigs in the absence (A) and presence (B) of ET-1 (10–7 mol/l). Platelets were coincubated with heart slices for 1 h at 37 °C and then recovered and incubation continued for 23 h. The bottom shows bar graphs representing the densitometric analysis of the Western blots. Results are represented as mean±S.E.M. of six different experiments. * P<0.05 with respect to controls. * P<0.05 with respect to ET-1.

 
Electrophoretic gel-shift mobility assays demonstrated induction of Sp-1 binding activity in nuclear extracts from ET-1-incubated heart slices (Fig. 7). This effect was enhanced by the presence of 109 platelets/well. Tirofiban (5x10–7 mol/l) prevented platelet-induced enhanced Sp-1 binding activity (Fig. 7). Greater doses of tirofiban (10–6 mol/l) failed to inhibit Sp-1 translocation more than 5x10–7 mol/l tirofiban (data not shown). Reaction specificity was demonstrated by the fact that excess (5x) unlabeled Sp-1 oligonucleotide prevented complex formation between nuclear extracts and labeled Sp-1 probe (Fig. 7).


Figure 7
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Representative electrophoretic mobility-shift assay (EMSA) showing the effect of ET-1 (10–7 mol/l) on Sp-1 translocation presence and in the absence of platelets (109 platelets/well) and platelets (109 platelets/well) plus tirofiban (5x10–7 mol/l). Nuclear extracts were prepared and subject to EMSA using a 32P-labelled Sp-1 oligonucleotide probe. Competition experiments with 5x excess (approximately 5 pmol) of unlabeled Sp-1 oligonucleotide and a positive control of nuclear extracts from HELA are also shown.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
Acknowledgments
 References
 
We have shown here that ET-1 induces expression of two pro-inflammatory proteins, COX-2 and tissue factor, in myocardium and in circulating leukocytes. Platelets, through GPIIb/IIIa receptor, participate in COX-2 and tissue factor induction elicited by ET-1 infusion.

Previous in vitro studies have suggested that ET-1 could be involved in inflammatory events. ET-1 induces leukocyte adhesion to cultured endothelial cells, stimulates cytokines release from isolated leukocytes and promotes vascular permeability [9,11,28]. We have now demonstrated that in vivo infusion of ET-1 stimulates expression of the proinflammatory markers COX-2 and tissue factor the myocardium and in circulating leukocytes of guinea-pigs.

Tissue factor is the primary activator of the extrinsic coagulation cascade and is rapidly induced in response to inflammatory stimuli such as cytokines [29]. Our data about induction of tissue factor expression by ET-1 correlate with observations showing that ET-1 caused in vivo coagulation in the microcirculation [30].

ET-1 infusion also increased expression of COX-2, the inducible isoform of COX which releases high amounts of inflammatory prostaglandins at the site of inflammation [15]. Sugimoto et al. and Hughes et al. have also reported that ET-1 induces COX-2 expression in glomerular mensagial cells [31,32], and McMillen et al. have recently demonstrated that ET-1 stimulated in vitro production of prostaglandin E2, a major product synthetized by COX-2 in human monocytes [33]. We observed here that, after ET-1 infusion, circulating leukocytes released higher amounts of prostacyclin and prostaglandin E2 which was associated with upregulation of COX-2 expression.

The ET-1 dose used did not produce significant blood pressure changes as in previous works [6,34,35], suggesting that modifications of tissue factor and COX-2 expression could not be attributable to changes in blood flow.

Platelet activation results in exposure and activation of GPIIb/IIIa receptors on the platelet surface. We and others have previously demonstrated that ET-1 induces platelet activation [6,7]. This is confirmed here by the fact that ET-1 infusion increased the number of platelets expressing activated GPIIb/IIIa. We thus then analyzed whether platelets could be involved in ET-1 mediated inflammatory response.

GPIIb/IIIa receptor blockade by tirofiban partially reduced ET-1-induced COX-2 and tissue factor expression in leukocytes and myocardium. These findings suggest that GPIIb/IIIa receptors on platelets participe in the proinflammatory action of ET-1.

Platelet activation could be a result rather than the mediator of ET-1-induced COX-2 expression. In this regard, Labonte et al. have showed that COX-2 ativity reduced ex vivo induced platelet activation after in vivo infusion of ET-1 in mice [36]. However, experiments in both thrombocytopenic guinea-pigs and in tirofiban-treated animals in which ET-1-induced COX-2 expression was reduced supported the hypothesis that platelets were involved in the upexpression of COX-2 by ET-1 with was further confirmed in the in vitro experiments. In this sense, Gawaz et al. have demonstrated that activated platelets stimulated the expression and release of inflammatory related proteins in cultured endothelium [37].

Although the tirofiban-dose used was enough to blunt ex vivo platelet activation, it only partially prevented COX-2 and tissue factor induction by ET-1 infusion. Moreover, thrombocytopenic guinea-pigs only partially prevented upexpression of COX-2 by ET-1 suggesting a direct effect of ET-1 in the induction of the inflammatory markers in the myocardium. Other authors have previously demonstrated that ET-1 directly induced COX-2 expression in isolated cultured cells [31,32].

The present study has focused on the effect of platelets on ET-1-related inflammation in the myocardium and leukocytes, but the underlying mechanisms remain to be elucidated. Platelets contain inflammatory mediators such as RANTES and interleukin-1 which are released from {alpha}-granules after platelet activation [38,39] and could be involved in the platelet-dependent ET-1-related inflammatory response. This hypothesis, is out of the scope of the present study and need further experiments. However, in order to assess whether platelets could induce intracellular responses associated to inflammation, we tested the activation of the transcription factor Sp-1 which has been involved in the expression of COX-2 and tissue factor [40,41]. ET-1 stimulated Sp-1 translocation in in vitro incubated heart slices which was enhanced by platelets and reduced by tirofiban, supporting that platelets throughout the GPIIb/IIIa receptors take part in the inflammatory response of the myocardium.

4.1 Pathophysiological and clinical implications
It has been recently reported that the blockade of GPIIb/IIIa receptors reduced infarct size in a canine model of ischemia-reperfusion independently of any effect on myocardial perfusion although the precise mechanisms remain unknown [42]. Since myocardial inflammation may contribute to cardiac myocyte loss, the here reported anti-inflammatory effect of GPIIb/IIIa blockade may be involved in this reduced infarct size. The concentration of ET-1 reached after 45 min infusion is about 20 pg/ml which is in a similar range to that measured in the plasma of unstable angina patients [43]. In this regard, Qiu et al. [44] have reported an acute increase in endothelin plasma levels during the acute phase of unstable angina suggesting a role for this peptide in the pathophysiology of this disease. Interestingly, the concept that the inflammatory process is not confined to a single vulnerable plaque but that it is a widespread acute inflammatory process in the endothelium of the coronary arterial bed has been recently suggested and is acquiring increasing importance [45]. Therefore, acute inflammatory endothelium-derived inflammatory mediators such as ET-1 may be involved in the inflammatory reaction of the acute coronary syndromes. Moreover, multiple fresh thrombi have been found in postmortem patients with unstable angina [46] that accordingly with our results could be related to the inflammatory reaction. Therefore, taken together, the here-reported model of acute cardiac ET-1 infusion may resemble an acute increase of ET-1 accompanied by platelet activation like it happens in the acute phase of unstable angina. Therefore, reduction of ET-1-induced myocardial inflammation via blockade of platelet GPIIb/IIIa receptors may have clinical consequences in the outcome of acute coronary syndromes and help to elucidate the therapeutic properties of GPIIb/IIIa blockers. However, the pathophysiologic role of endogenous ET-1 in the setting of the inflammatory myocardial reaction of unstable angina is likely to be clarified with the evaluation of selective ET-1 antagonists which warrants further studies.

In summary, the present results demonstrated that ET-1 stimulated the in vivo expression of the proinflammatory proteins COX-2 and tissue factor in the myocardium and in leukocytes and the involvement of GPIIb/IIIa platelet receptors. Our findings also suggested for the first time an antiinflammatory effect of the GPIIb/IIIa blocker tirofiban that could extend its antithrombotic effect.

Time for primary review 21 days.


   Acknowledgments
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
 Acknowledgments
References
 
This work was supported by a school-grant from Merck Laboratories and from Sociedad Española de Cardiologia (SAF 2000/0024). L. Molero, A. García Mendez and P. Jiménez Mateos-Caceres are fellows from Fundación Conchita Rábago. We thank Begoña Larrea for secretarial assistance.


   References
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 

  1. Phillips D.R., Charo I.F., Parise L.V., Fitgerald L.A. The platelet membrane glycoprotein IIb/IIIa complex. Blood (1988) 71:841–843.
  2. Leung L., Nachman R. Molecular mechanisms of platelet aggregation. Ann. Rev. Med. (1986) 37:179–186.[CrossRef][Web of Science][Medline]
  3. Haas T.A., Plow E.T. The cytoplasmic domain of {alpha}IIbβ: a ternary complex of the integrin A and B subunit and a divalent cation. J. Biol. Chem. (1996) 271:6017–6026.[Abstract/Free Full Text]
  4. The CAPTURE Study Group. Randomised placebo-controlled trial of abciximab before and during coronary intervention in refractory unstable angina. Lancet (1997) 349:1429–1435. The CAPURE study.[CrossRef][Web of Science][Medline]
  5. PRISM-PLUS study Investigators. Inhibition of the platelet glycoprotein IIb/IIIa receptor with tirofiban in unstable angina and non-Q-wave myocardial infaction. N. Engl. J. Med. (1998) 338:1488–1497.[Abstract/Free Full Text]
  6. Lopez-Farré A., Caramelo C Esteban A., Alberola M.L., Millas I Monton M., Casado S. Effect of aspirin on platelet neutrophil interactions. Role of nitric oxide and endothelin-1. Circulation (1995) 91:2080–2088.[Abstract/Free Full Text]
  7. Halim A., Kanayama N., el Maradny E., Machara K., Terao T. Endothelin-1 evoked an increase and oscillations in cytosolic calcium concentration in adherent single human platelet and increased GMP-140 (P-selectin) in platelet suspension. Thromb. Res. (1995) 80:105–112.[CrossRef][Web of Science][Medline]
  8. Yanagisawa M., Kuriraba H., Kimura S., Tomobe Y., Kobayashi M., Mitsui Y., Goto K., Masoki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature (1988) 332:411–415.[CrossRef][Medline]
  9. López-Farré A., Riesco A., Espinosa G., Digiuni E., Cernadas M.R., Alvarez V., Montón M., Rivas F., Gallego M.J., Egido J., Casado S., Caramelo C. Effect of endothelin-1 on neutrophil adhesion to endothelial cells and perfused heart. Circulation (1993) 88:1166–1171.[Abstract/Free Full Text]
  10. McCarron R.M., Wang L., Stanimirovic D.B., Spatz M. Endothelin induction of adhesion molecule expression on human brain microvascular endothelial cells. Neurosci. Lett. (1993) 156:31–34.[CrossRef][Web of Science][Medline]
  11. McMillen M.A., Huribal M., Cunningham M.E., Kumar R., Sumpio B.E. Endothelin-1 increases intracellular calcium in human monocytes and causes production of interleukin-6. Crit. Care Med. (1995) 23:34–40.[CrossRef][Web of Science][Medline]
  12. Lerma M.A., Edwards B.S., Hallet J.N., Henblein D.M., Sandberg S.M., Burnet J.C. Circulating and tissue endothelium immunoreactivity in advanced atherosclerosis. N. Engl. J. Med. (1991) 325:997–1001.[Abstract]
  13. Bath P.M., Martin J.F. Serum platelet-derived growth factor and endothelin concentration in human hypercholesterolemia. J. Int. Med. (1991) 230:313–317.[Web of Science][Medline]
  14. Margulies K.B., Hildebrand F.L., Lerman A., Percella M.A., Burnett J.C. Increased endothelin in experimental heart failure. Circulation (1990) 82:2226–2230.[Abstract/Free Full Text]
  15. Hla T., Bishop-Bailey D., Lin C.H., Schaefers H.J., Trifan O.C. Cyclooxigenase-1 and -2 isoenzymes. Int. J. Biochem. Cell Biol. (1999) 31:551–557.[CrossRef][Web of Science][Medline]
  16. Cunningham M.A., Romas P., Hutchinson P., Holdsworth S.R., Tipping P.G. Tissue factor and factor VIIa receptor/ligand interactions induce proinflammatory effects in macrophages. Blood (1999) 94:3413–3420.[Abstract/Free Full Text]
  17. Lynch J.J., Cook J.J., Sitko G.R., Holahan M.A., Ramjit D.R., Mellot M.J., Stranieri M.T., Stabilito I.I., Zhang G., Lynch R.J. Non-peptidic glycoprotein IIb/IIIa inhibitors. Antithrombotic effects of MK-0383. J. Pharmacol. Exp. Ther. (1995) 272:20–32.[Abstract/Free Full Text]
  18. Barrett J.S., Murphy G., Peerlinck K., De Lepeleire I., Gould R.J., Panebianco D., Hsnd E., Deckmyn H., Vermylen J., Arnout J. Pharmacokinetics and pharmacodynamics of MK-383, a selective non-peptide platelet glycoprotein-IIb/IIIa receptor antagonist, in healthy men. Clin. Pharmacol. Ther. (1994) 56:377–384.[Web of Science][Medline]
  19. Laemmli N.K. Change of structural proteins during the assembly of the head of bacteriophage T4. Nature (1970) 227:680–685.[CrossRef][Medline]
  20. de Frutos T., Sanchez de Miguel L., Farré J., Romero J., Marcos-Alberca P., Nuñez A., Rico L., López-Farré A. Expression of an endothelial-type nitric oxide synthase isoform in human neutrophils: modification by tumor necrosis factor-alpha and during myocardial infarction. J. Am. Coll. Cardiol. (2001) 37:800–807.[Abstract/Free Full Text]
  21. Alonso J., Sanchez de Miguel L., Montón M., Casado S., Lopez-Farré A. Endothelial cytosolic proteins bind to the 3'-untranslated region of endothelial nitric oxide synthase mRNA: regulation by tumor necrosis factor alpha. Mol. Cell. Biol. (1997) 17:5719–5726.[Abstract]
  22. López-Farré A., Riesco A., Digimi E., Mosquera J.R., Caramelo C., de Miguel L.S., Millás I., de Frutos T., Cernadas M.R., Montón M., Alonso J., Casado S. Aspirin-stimulated nitric oxide production by neutrophils after acute myocardial ischemia in rabbits. Circulation (1996) 94:83–87.[Abstract/Free Full Text]
  23. Gonzalez-Fernandez F., López-Farré A., Rodriguez Feo J.A., Farré J., Guerra J., Fortes J., Millas I., Garcia-Duran M., Rico L., Sanchez de Miguel L., Casado S. Expression of inducible nitric oxide synthase after endothelial denudation of the rat carotid artery. Role of platelets. Circ. Res. (1998) 83:1080–1087.[Abstract/Free Full Text]
  24. Jimenez A., Arriero M.M., López-Blaya A., Gonzalez-Fernandez F., García R., Fortes J., Millas I., Velasco S., Sanchez de Miguel L., Rico L., Farré J., Casado S., López-Farré A. Regulation of endothelial nitric oxide synthase expression in the vascular wall and in mononuclear cells from hypercholesterolemic rabbits. Circulation (2001) 104:1822–1830.[Abstract/Free Full Text]
  25. López-Farré A., Bernabeu F., Gomez-Garre D., Ramon y Cajal S., Braquet P., López-Novoa J.M. Platelet activating factor antagonist treatment protects against postischemic acute renal failure in rats. J. Pharmacol. Exp. Ther. (1990) 253:328–333.[Abstract/Free Full Text]
  26. Ziegler-Heitbrock H.W.L., Sternsdonf T., Liese J., Belohradsky B., Weber C., Wedel A., Schreck R., Baverle P., Strobel M. Pyrrolidine dithiocarbamate inhibits NF-KB mobilization and TNF production in human monocytes. J. Immunol. (1993) 151:6986–6993.[Abstract]
  27. Sanchez de Miguel L., de Frutos T., Gonzalez-Fernandez F., del Pozo V., Lahoz C., Jimenez A., Rico L., García R., Aceituno E., Millás I., Gomez J., Farré J., Casado S., Lopez-Farre A. Aspirin inhibits inducible nitric oxide synthase expression and tumor necrosis factor-{alpha} release by cultured smooth muscle cells. Eur. J. Clin. Invest. (1999) 29:93–99.[CrossRef][Web of Science][Medline]
  28. Filep J.G., Földer-Filep E., Rousseau A., Fournier A., Sirois P., Yano M. Endothelin-1 enhances vascular permeability in the rat heart though the ETA receptor. Eur. J. Pharmacol. (1992) 219:343–344.[CrossRef][Web of Science][Medline]
  29. Mackman N. Regulation of the tissue factor gene. FASEB J. (1995) 9:883–889.[Abstract]
  30. Halim A., Kanayama N., el Maradny E., Machara K., Terao T. Coagulation in vivo microcirculation and in vitro caused by endothelin-1. Thromb. Res. (1993) 72:203–209.[CrossRef][Web of Science][Medline]
  31. Sugimoto T., Haneda M., Sawano H., Isshiki K., Maeda S., Koya D., Inoki K., Yasuda H., Kashiwagi A., Kikkawa R. Endothelin-1 induces cyclooxygenase-2 expression via nuclear factor of activated T-cell transcription factor in glomerular mesangial cell. J. Am. Soc. Nephrol. (2001) 12:1359–1368.[Abstract/Free Full Text]
  32. Hughes A., Padilla E., Kutchera W., Michael J.R., Kohan D.E. Endothelin-1 induction of cyclooxygenase-2 expression in rat mesangial cells. Kidney Int. (1995) 47:53–61.[Web of Science][Medline]
  33. McMillen M.A., Huribal M., Kumar R., Sumpio B.E. Endothelin-stimulated human monocytes produce prostaglandin E2 but not leukotriene B4. J. Surg. Res. (1993) 54:331–335.[CrossRef][Web of Science][Medline]
  34. Espinosa G., López-Farré A., Cernadas M.R., Manzarbertia F., Tan D., Digiuni E., Mosquera J.R., Montón M., Millas I., Hernando L., Casado S., Caramelo C. Role of endothelin in the pathophysiology of renal ischemia-reperfusion in normal rabbits. Kidney Int. (1996) 50:776–782.[Web of Science][Medline]
  35. Clozel J.P., Clozel M. Effects of endothelin in the coronary vascular bed in open-chest dogs. Circ. Res. (1989) 65:1193–1200.[Abstract/Free Full Text]
  36. Labonte J., Brochu I., Honore J.C., D'Orleans-Juste P. Role of ETB and B2 receptors in the ex vivo platelet inhibitory properties of endothelin and bradykinin in the mouse. Br. J. Pharmacol. (2001) 132:S184–S186.
  37. Gawaz M., Newmann F.J., Dickfeld T., Koch W., Laugwitz K.L., Adelsberger H., Langenbrink K., Page S., Neumeier D., Schoming A., Brand K. Activated platelets induce monocyte chemotactic protein-1 secretion and surface expression of intracellular adhesion molecule-1 on endothelial cells. Circulation (1998) 98:1164–1171.[Abstract/Free Full Text]
  38. Hawrylowing C.M., Santoro S.A., Platt F.M., Uname E.R. Activated platelets express IL-1 activity. J. Immunol. (1989) 143:4015–4018.[Abstract]
  39. Kameyoshi Y., Dorschner A., Mallet A.L., Christophers E., Schroder J.M. Cytokine RANTES released by thrombin-stimulated platelets is a potent attractant from human eosinophils. J. Exp. Med. (1992) 176:587–592.[Abstract/Free Full Text]
  40. Hla T., Bishop-Bailey D., Lin C.H., Schaefers H.J., Trifan O.C. Cyclooxygenase-1 and -2 isoenzymes. Int. J. Biochem. Cell Biol. (1999) 31:551–557.[CrossRef][Web of Science][Medline]
  41. Krikun G., Schatz F., Mackman N., Guller S., Demopoulos R., Lockwood C.J. Regulation of tissue factor gene expression in human endometrium by transcription factors Sp1 and Sp3. Mol. Endocrinol. (2000) 14:393–400.[Abstract/Free Full Text]
  42. Knigma J.G., Plante S., Bogaty P. Platelet GPIIb/IIIa receptor blockade reduces infarct size in a canine model of ischemia-reperfusion. J. Am. Coll. Cardiol. (2000) 36:2317–2324.[Abstract/Free Full Text]
  43. Dupuis J., Tardif J.C., Cernacek P., Theroux P. Cholesterol reduction rapidly improves endothelial function after acute coronary syndromes. The RECIFE (reduction of cholesterol in ischemia and function of the endothelium) trial. Circulation (1999) 99:3227–3233.[Abstract/Free Full Text]
  44. Qiu S., Theroux P., Marcil M., Solynoss B.C. Plasma endothelin-1 levels in stable and unstable angina. Cardiology (1993) 82:12–19.[Web of Science][Medline]
  45. Buffor A., Biasucci L.M., Liuzzo G., Dónofilo G., Crea F., Maseri A. Widespread coronary inflammation in unstable angina. N. Engl. J. Med. (2002) 347:5–12.[Abstract/Free Full Text]
  46. Falk E., Sah P.K., Fuster V. Coronary plaque disruption. Circulation (1995) 92:657–671.[Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Molero, L.
Right arrow Articles by López-Farré, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Molero, L.
Right arrow Articles by López-Farré, A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?