Cardiovascular Research

Cardiovascular Research 2001 49(1):189-199; doi:10.1016/S0008-6363(00)00220-0
© 2001 by European Society of Cardiology

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Copyright © 2000, European Society of Cardiology

Transient interaction of activated platelets with endothelial cells induces expression of monocyte-chemoattractant protein-1 via a p38 mitogen-activated protein kinase mediated pathway

Implications for atherogenesis

Timm Dickfeld^a, Ernst Lengyel^c, Andreas E May^b, Steffen Massberg^b, Korbinian Brand^d, Sharon Page^d, Christiane Thielen^b, Kirsten Langenbrink^b and Meinrad Gawaz^b,*

^aMedical Department, Duke University, Durham, NC, USA
^b1. Medizinische Klinik Klinikum rechts der Isar and Deutsches Herzzentrum, Technische Universität, Lazarettstrasse 36, 80636 Munich, Germany
^cFrauenklinik Technische Universität, Munich, Germany
^dInstitut für Klinische Chemie and Pathobiochemie, Technische Universität, Munich, Germany

^* Corresponding author. Tel.: +49-89-1218-4012; fax: +49-89-1218-4003 gawaz{at}dhm.mhn.de

Received 10 May 2000; accepted 23 August 2000

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: Activated platelets induce alterations of chemotactic and adhesive properties of endothelial cells, a critical initial step in atherogenesis. We investigated the effect of transient interaction of activated platelets with cultured human umbilical vein endothelial cells (HUVECs) on secretion of monocyte chemoattractant protein-1 (MCP-1), a key molecule in monocyte chemotaxis and transmigration. Methods and results: Transient interaction of -thrombin-activated platelets with endothelial cells for 10–120 min substantially induced endothelial secretion of MCP-1, monocyte chemotaxis and adhesion to HUVECs. Platelet-induced secretion of MCP-1 and monocyte–endothelium adhesion was reduced by the MAP kinase p38-specific inhibitor SB203580, but not by other kinase inhibitors including PD98059, wortmannin, or rapamycin. In addition, activated platelets induced transcription of a luciferase reporter construct containing a MCP-1 promotor, an effect that could be inhibited by SB203580. Overexpression of dominant-negative mutants of MAP kinase p38, CSBP2-(D168A) and CSBP2-(T180E,Y182E) reduced platelet-induced expression of MCP-1. Conclusions: Activation of the p38 MAP kinase and consecutive endothelial secretion of MCP-1 induced through transient interaction of activated platelets might play an important role in atherogenesis.

KEYWORDS Platelets; Endothelial function; Atherosclerosis; Complement activation

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Although originally envisioned as passive and inert, the intact endothelium has been shown to allow adhesion of activated, but not of resting platelets both in vitro [1,2] and in vivo [3]. Interaction of activated platelets with the endothelium and consecutive inflammatory responses within the vessel wall might contribute substantially to early steps of atherosclerosis [4]. Activated platelets release a variety of proinflammatory mediators into their microenvironment, such as growth factors and cytokines [4]. Recently, several platelet-derived substances such as IL-1β [5,6] and CD40L [7] released from activated platelets have been shown to induce a variety of inflammatory genes including the monocyte chemoattractant protein-1 (MCP-1) [8]. MCP-1 is a member of the C–C chemokine family and attracts blood monocytes to inflammatory sites [9]. MCP-1 is expressed by a variety of cell types including endothelial cells in response to several stimuli [9]. Chemotaxis and transmigration of circulating monocytes through the endothelial surface is a prerequisite for monocyte–macrophage transformation — a mechanism involved in early steps of atherosclerosis [4].

Endothelial cells have been shown to produce MCP-1 in response to extracellular stimuli such as cytokines as well as thrombin [10]. MCP-1 gene expression in human endothelial cells involves activation of transcription factor nuclear factor B (NF-B) [11–13] and the MKK6/p38 stress kinase cascade [14]. Recently, we showed that activated platelets induce MCP-1 gene expression on a transcriptional level involving activation of NF-B [6,8].

The purpose of the present study was to evaluate the effect of transient platelet–endothelium interaction on endothelial MCP-1 expression, monocyte chemotaxis to endothelium, and to characterize signal transduction events involved in these processes.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Cell culture
Primary cultures of human umbilical vein endothelial cells (HUVECs) were obtained by collagenase treatment of umbilical cord veins as described recently [6]. Cells from four to six preparations were pooled and cultivated in 24-well culture plates (NUNC) in complete endothelium cell growth medium (PromoCell), containing 2% fetal bovine serum (FCS), 1 µg/ml hydrocortisone, 0.1 ng/ml hEGF, 1.0 ng/ml FGF, 50 µg/ml gentamycin, 2.5 µg/ml amphotericin B. The human myelo-monocytic HL60 cell line was cultured in RPMI 1640 medium supplemented with 10% FCS, 1% sodium pyruvate, 1 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco-BRL).

2.2 Coculture of endothelial monolayers with platelets
Platelets were isolated from acid-citrate–dextrose-anticoagulated whole blood collected from healthy non-smoking individuals that were not on any medication known to affect platelet function [6]. Platelets were washed and resuspended in Tyrode's solution–HEPES buffer (HEPES 2.5 mmol/l, NaCl 150 mmol/l, NaHCO₃ 12 mmol/l, KCl 2.5 mmol/l, MgCl₂ 1 mmol/l, CaCl₂ 2 mmol/l, D-glucose 5.5 mmol/l and 1 mg/ml BSA, pH 7.4) to obtain a platelet count of 4x10⁸/ml. Platelets were prestimulated with 2 U/ml -thrombin (Sigma, Munich, Germany) for 20 min followed by antagonization of thrombin by 5 U/ml hirudin (Roche). Activation of -thrombin-stimulated platelets was verified by determination of surface expression of P-selectin (mean intensity of CD62 P-immunofluorescence (mean±standard deviation (n = 4): 168±35 versus 241±60, nonstimulated versus -thrombin-stimulated platelets) [15]. Thereafter, 250 µl of the platelet suspension were added to 250 µl complete endothelium cell growth medium (final platelet count 2x10⁸/ml) and transferred to wells of 24-well plates covered with confluent monolayers of HUVECs. HUVECs were left untreated or were incubated for 10, 30, 120 min or 5 h at 37°C with activated platelets. Then, platelets were removed from HUVECs through multiple gentle washing steps and 500 µl of culture medium was added to the wells until a total incubation time of 5 h was completed. In preliminary studies we ensured by visual microscopy and flow cytometry using a platelet-specific anti-CD42b mAb that after the washing steps, virtually all platelets were removed from the endothelial monolayer. MCP-1 secretion was evaluated by ELISA measurements of the supernatant (Quantokine R&D). In control experiments, HUVECs were stimulated with 100 pg/ml rhIL-1β as indicated. In some experiments HUVECs were pretreated with the inhibitors SB203580 (10 µg/ml) (inhibits p38 MAPK), PD98059 (10 µg/ml) (inhibits Erk1/2 MAPK), wortmannin (100 nM) (inhibitor of PI-3 kinase), or rapamycin (20 ng/ml) (inhibits Akt-p60-S6 kinase) 60 min prior to activation (all from Calbiochem). In some experiments, HUVECs were incubated with membranes (1 mg/ml) isolated from nonstimulated or -thrombin (2 U/ml)-activated platelets as described for 60 min and MCP-1 secretion was determined in the supernatant after completion of 5 h [8,16]. Platelet releasate was obtained by removal of the supernatant after centrifugation of suspensions of nonstimulated or -thrombin (2 U/ml)-activated platelets (2x10⁸/ml) [8,16].

2.3 MCP-1 ELISA, flow cytometry and endotoxin assay
The supernatant of cultured HUVECs treated with platelets or agonists was aspirated, centrifuged at 2700 g for 10 min, and stored at –80°C. Concentrations of MCP-1 protein were determined by use of an ELISA (Biermann, Germany) with a detection limit of 5 pg/ml. In some experiments with transiently transfected HUVECs MCP-1 generation was determined by flow cytometry as described [14] using an anti-MCP-1 polyclonal antibody (Biermann). To eliminate endotoxin contamination, all crystalloid solutions were ultrafiltered (U2000, Gambro) and stock solutions of proteins were decontaminated by polymyxin columns (Pierce). To exclude endotoxin contamination, all cell suspensions at the end of each experiments were evaluated by chromogenic limulus amoebocyte lysate assay (Schulz).

2.4 Transient transfection procedures and luciferase reporter analysis
HUVECs were cultured to 70% confluence before transient transfection using 2 µg DNA/24-well of the Flag epitope-tagged dominant-negative p38 constructs (pCDN-CSBP2 (D168A), pCDN-CSBP2 (T180E,Y182E)) [17,18] (kindly provided by Smith Kline, King of Prussia), or the control vector plasmid pCDN and a polycationic transfection reagent (SuperFect^® Qiagen, Hilden, Germany) [8]. After 24 h, transfected HUVECs were stimulated with -thrombin-activated platelets or rhIL-1β as described above and MCP-1 secretion by HUVECs was evaluated. Transgene protein expression in HUVECs was verified by immunoblotting using the polyclonal anti-p38 MAP kinase antibody (1/500, Santa Cruz Biotechnology, Heidelberg, Germany) that detects both the non- and phosphorylated form of p38 and an anti-flag monoclonal antibody (Stratagene, clone M2). The MCP-1-luciferase reporter construct contained the proximal promotor region and distal enhancer region of the human MCP-1 gene [19,20] and was kindly provided by Dr. Teizo Yoshimura (NIH, Frederick, MD, USA). The MCP-1 reporter construct was cotransfected with a Renilla luciferase control plasmid (pRL-TK, Promega) [8]. Cells were incubated with rhIL-1β, platelets, or inhibitors as indicated 24 h after endothelial transfection. After 5 h the luciferase activity was measured according to the dual luciferase reporter assay system (Promega).

2.5 Monocyte chemotaxis
To evaluate the effect of platelet-induced endothelial activation on chemotaxis of HL60 cells, a trans-well culture system was used [6]. In brief, HUVECs were cultivated in 24-well culture plates, left untreated or pretreated with SB203580 (10 µg/ml) or PD98059 (10 µg/ml) for 60 min and stimulated with -thrombin-activated platelets or rhIL-1β for a further 60 min. Myelo-monocytic HL60 cells (2x10⁶/ml) were added on top using trans-well culture inserts (8.0 µm pore size, Falcon) that allowed physical separation of HL60 from the endothelial monolayer. After an incubation time of 5 h transmigration of HL60 cells through the filter was evaluated by cell counting [21].

2.6 p38 MAP kinase assay and immunoblotting
HUVECs were washed once in phosphate-buffered saline (PBS) (Promega, Germany) and lysed using a buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 1 mM sodium-ortho-vanadate, 1 mm PMSF, 10 µg/ml aprotinin (all obtained from Sigma) at 4°C for 10 min. Samples were centrifuged at 8000 g for 10 min and equal amounts of protein incubated with 30 µl protein A agarose (Boehringer Mannheim, Germany) and 0.5 µg/ml polyclonal anti-p38 MAP kinase antibody (Santa Cruz) for 3 h at 4°C. After a two-step washing procedure with buffer A (20 mM HEPES, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 10 mM pyrophosphate) followed by kinase buffer B (20 mM HEPES, 10 mM MgCl₂, 400 µM sodium-ortho-vanadate, 1 mM DTT), each sample was incubated with 40 µl kinase buffer supplemented with 2 µg of ATF2 (Santa Cruz), 50 µM unlabeled ATP and 10 µCi [³²P]-ATP (ICN Biomedicals, Eschwege, Germany) for 30 min at 30°C. The reaction was stopped by adding Lämmli sample buffer and phosphorylated ATF2 resolved on a 12.5% SDS–PAGE and visualized by autoradiography. Aliquots of proteins were probed by immunoblotting with a polyclonal anti-p38 MAP kinase antibody (1/500, Santa Cruz Biotechnology) that detects both the non- and phosphorylated form of p38.

2.7 Confocal laser immunofluorescence microscopy
HUVECs were grown on coverslips coated with 5 µg/ml vitronectin (Sigma) at 4°C overnight and blocked for 1 h with 5% BSA in PBS at room temperature. Stimulated or nonstimulated HUVECs were fixed with 2% formaldehyde in PBS for 30 min at room temperature, washed twice with 2% glycine in PBS, permeabilized with 0.2% Triton X-100, and stained with polyclonal anti-p38 MAP kinase antibody (clone C-20) or phosphospecific p38 antibody (clone SC-7937, Santa Cruz Biotechnology) for 60 min.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Transient incubation of endothelial cells with thrombin-activated platelets induces secretion of MCP-1, monocyte chemotaxis, and monocyte adhesion to HUVECs
Confluent monolayers of HUVECs were left untreated or were incubated for 10, 30, 60, 120 min and 5 h with -thrombin-activated platelets or with 100 pg/ml rhIL-1β. Platelets or rhIL-1β were removed from the endothelial monolayer and the amount of MCP-1 in the supernatant was determined 5 h subsequent to activation. Transient incubation of HUVECs with -thrombin-activated platelets for 30 min resulted in a substantial increase of MCP-1 secretion comparable to rhIL-1β stimulation (Fig. 1A). The time course of platelet-induced endothelial secretion of MCP-1 was similar to the rhIL-1β-induced MCP-1 expression (Fig. 1A). In contrast, incubation of HUVECs with quiescent platelets did not induce substantial MCP-1 secretion (Fig. 1A). In preliminary control experiments we found that incubation of HUVECs with -thrombin plus hirudin did not result in substantial secretion of MCP-1 (Fig. 1B). Similarly, secretion of MCP-1 was enhanced in the presence of supernatant of -thrombin-activated platelets compared with supernatant derived from nonstimulated platelets (Fig. 1B) (P<0.01). No significant change in MCP-1 secretion was found in the presence of membranes isolated from donor -thrombin-activated platelets (Fig. 1B).

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Effect of transient interaction of activated platelets with HUVECs. (A) HUVECs were left untreated or were incubated with -thrombin-activated platelets (2x108/ml) for 10, 30, 60, 120 and 300 min and thereafter, platelets were removed by culture medium. After completion of a total 5 h incubation time MCP-1 concentration was determined in the supernatant. rhIL-1β (100 pg/ml) or quiescent platelets were used to activate HUVECs in control experiments. Depicted are mean and SEM of four independent experiments performed in triplicate. *, Significant differences compared to baseline values (P<0.01). (B) HUVECs were incubated with nonstimulated or -thrombin-activated platelets (2x108/ml), membranes, or releasates for 60 min and secretion of MCP-1 was evaluated after 5 h. As control, secretion of MCP-1 of untreated or HUVECs treated with -thrombin (2 U/ml) plus hirudin (5 U/ml) for 60 min are shown. Depicted are mean±SEM of three independent experiments.

 
Next, we evaluated the functional relevance of platelet-induced endothelial MCP-1 production with respect to monocyte chemotaxis. Monocyte migration towards endothelial cells pretreated with -thrombin-activated platelets was approximately 2.5-fold increased compared to unstimulated HUVECs (P<0.01) (Fig. 2); that slightly exceeded the migration observed with rhIL-1β (100 pg/ml)-stimulated HUVECs (Fig. 2).

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Effect of platelet-induced HUVEC activation on monocyte chemotaxis to HUVECs. HUVECs were preincubated with -thrombin-activated platelets or with 100 pg/ml rhIL-1β for 60 min. Thereafter, chemotaxis of monocytic HL60 cells was evaluated using trans-well culture inserts that allows physical separation of both cell types (modified Boyden chamber). Depicted are mean and SEM of three independent experiments performed in duplicate. *, Significant differences compared to baseline values (P<0.01).

 
3.2 p38 inhibitor, SB203580, inhibits platelet-induced endothelial secretion of MCP-1, chemotaxis of monocytes
After pretreatment of HUVECs for 60 min with the kinase inhibitors as indicated, activated platelets or rhIL-1β were added for 60 min to the cells, removed, and after further incubation for 4 h MCP-1 was determined in the cell supernatant. SB203580 exerted a significant reduction in platelet-induced MCP-1 secretion by 40–50% compared to untreated endothelial cells (P<0.01) (Fig. 3). The other inhibitors did not significantly affect the platelet-induced production of MCP-1 (Fig. 3).

View larger version (39K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effect of kinase inhibitors on platelet-induced secretion of MCP-1. Confluent monolayers of HUVECs were pretreated (60 min) with SB203580 (10 µg/ml), PD98059 (10 µg/ml), rapamycin (100 nM), or wortmannin (20 ng/ml) and cells were then incubated with -thrombin-activated platelets for 60 min. MCP-1 secretion was determined after 4 h as described above. Depicted are mean±SEM of three independent experiments. *, Significant differences compared with baseline values (P<0.01).

 
To assess the effect of the p38 inhibitor SB203580 in functional aspects, HUVECs were pretreated with inhibitors and platelet-induced monocyte chemotaxis was evaluated as described above. Similar to the observed inhibition of MCP-1 secretion, preincubation of HUVECs with SB203580 but not with PD98059 (Fig. 4) or with the other tested inhibitors (data not shown) resulted in approximately 50% reduction of platelet-induced chemotaxis of monocytes to endothelial cells (Fig. 4).

View larger version (27K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Effect of pretreatment of HUVECs with inhibitor SB203580 and PD98059 on platelet-induced monocyte chemotaxis to HUVECs. Confluent monolayers of HUVECs were pretreated with SB203580 (10 µg/ml) or PD98059 (10 µg/ml), and cells were stimulated with -thrombin-activated platelets for 60 min. Chemotaxis of HL60 cells to HUVECs were then evaluated as described above. The results (mean±SEM) of three experiments are shown. *, Significant differences compared to baseline values (P<0.01).

 
3.3 Activated platelets induce p38 MAP kinase activation, nuclear translocation of p38, and MCP-1 promotor dependent transcription
p38 MAPK is activated by dual phosphorylation of tyrosine and threonine [22], and the detection of p38-phosphorylation has been widely used to measure the degree of p38 activation [23]. To study activation of endogenous p38 by activated platelets, HUVECs were exposed for various times to -thrombin-activated platelets or rhIL-1β, and p38 kinase activation determined in an immunocomplex kinase assay. Exposure of HUVECs to activated platelets induced activation of p38 within 15 min which decreased after 60 min (Fig. 5A). When HUVECs were stimulated with rhIL-1β, p38 kinase activity increased, albeit to a lesser extent (Fig. 5A). As controls, incubation of HUVECs with -thrombin (2 U/ml) plus hirudin (5 U/ml) for 15 min did not result in substantial activation of p38 (Fig. 5B). The observed enhanced phosphorylation was not due to a variation in total amount of p38 MAPK because the strength of the p38-immunoreactive band was not significantly different (Fig. 5).

View larger version (42K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Time course of p38 MAP kinase activation induced through -thrombin-activated platelets. Confluent monolayers of HUVECs were stimulated with -thrombin-activated platelets or rhIL-1β for the indicated time and activation of p38 was determined in immunoprecipitated samples by autoradiographic detection of phosphorylated ATF2 as described in Methods (upper panel) (A). Aliquots were probed for total amount of p38 protein by immunoblotting (lower panel) (A). (B) As control, HUVECs were treated with -thrombin (2 U/ml) plus hirudin (5 U/ml) or rhIL-1β (100 pg/ml) for 15 min and p38 activation was determined.

 
Members of the MAPK family are translocated to the nucleus upon activation where they elicit their effects on gene regulation [23,24]. Immunofluorescence staining using an antibody recognizing the phosphorylated form of p38 demonstrated that in the absence of activated platelets, virtually no phosphorylated p38 MAPK was detectable (Fig. 6). Stimulation of the cells with activated platelets for 15 min resulted in enhanced immunoreactivity to phosphorylated p38 MAPK both in the cytoplasma and in the nucleus (Fig. 6).

View larger version (79K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Immunofluorescence of platelet-induced activation of p38 and nuclear translocation. HUVECs were treated with -thrombin-activated platelets or rhIL-1β for 15 min and subcellular distribution of p38 was detected using phospho-p38-specific or pan-p38 mAb. Results are representative of three independent experiments (x280) (A). (B) Magnification x420. Arrows indicate translocation to the nucleus.

 
To analyse transcriptional regulation of the MCP-1 promoter by the p38 pathway, HUVECs were transfected with a MCP-1 promotor/enhancer luciferase construct containing the enhancer (between –2742 and –2513) and promotor regions (between –107 and +60) of the human MCP-1 promotor [19,20]. At 24 h after transfection HUVECs were left untreated or were pretreated with 10 µg/ml SB203580 for 60 min before stimulation with -thrombin-activated platelets (Fig. 7). The p38 inhibitor SB203580 inhibited MCP-1 promotor activity by 50% (Fig. 7).

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Effect of p38 inhibitor on platelet and rhIL-1β-induced MCP-1 promotor activity in HUVECs. HUVECs were cotransfected with a MCP-1 promotor/enhancer luciferase construct and pRL-TK Renilla luciferase control plasmid. After 24 h, cells were left untreated or were pretreated with 10 µg/ml SB203580 for 60 min. HUVECs were stimulated with -thrombin-activated platelets or with rhIL-1β for the indicated time. After a total incubation time of 5 h generation of luciferase was evaluated. The results of four experiments are shown. *, Significant differences (P<0.01) between groups.

 
3.4 Overexpression of dominant-negative p38 MAPK mutants reduces platelet-induced MCP-1 secretion
Dominant-negative mutants of p38 have been shown to inhibit activation of the p38 MAP kinase cascade [14] and MCP-1 promotor-dependent transcription in endothelial cells [14]. Subconfluent monolayers of HUVECs were cotransfected with the p38 mutants CSBP2 (D168A), CSBP2 (T180E,Y182E) or the control vector pCDN with the MCP-1 promotor/enhancer luciferase construct. Expression of flag-tagged p38 mutants in HUVECs was verified by immunoblotting using anti-p38 and anti-flag antibodies (Fig. 8A). Overexpression of both p38 mutants results in substantial reduced transcription of MCP-1 by approximately 50% (P<0.01) (Fig. 8B). In addition, secretion of MCP-1 protein induced through transient interaction of HUVECs with -thrombin-activated platelets was decreased in HUVECs transfected with p38 mutants compared to the vector control (P<0.01) (Fig. 9).

View larger version (35K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 Inhibition of MCP-1 promotor transcription by dominant negative p38 MAP kinase mutants. (A) Immunoblot of HUVECs transiently transfected with dominant negative mutants of p38 CSBP2 (D168A), CSBP2 (T180E, Y182E), or the control plasmid pCDN. (B) For MCP-1 promotor transcription analysis cells were cotransfected with the p38 mutants and luciferase expression under the control of the MCP-1 promotor was determined. The results of four independent experiments are shown. *, Significant differences (P<0.01) compared to baseline.

 

View larger version (30K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 9 Effect of dominant-negative p38 mutants on platelet-induced generation of MCP-1. For evaluation of MCP-1 protein generation HUVECs were transfected with the p38 mutants CSBP2 (D168A), CSBP2 (T180E, Y182E), or the control plasmid pCDN as described above. Thereafter, -thrombin-activated platelets were added for 60 min to the transfected HUVECs and after further 4 h endothelial generation of MCP-1 was determined by flow cytometry. The results of four independent experiments are shown. *, Significant differences (P<0.01) compared to control vector.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The major findings of the present paper are: (1) transient interaction of -thrombin-activated platelets with the endothelium results in substantial secretion of MCP-1 and subsequent chemotaxis of monocytes; (2) -thrombin-activated platelets induce activation of the MAP kinase p38, nuclear translocation of p38, and MCP-1 promotor dependent transcription; (3) the p38 inhibitor SB203580 substantially reduces platelet-induced endothelial secretion of MCP-1, chemotaxis and adhesion of monocytes to endothelial cells and (4) overexpression of the dominant-negative p38 mutants inhibits platelet-induced MCP-1 promotor dependent transcription and endothelial secretion of MCP-1.

These findings imply that transient interaction of activated platelets with the endothelium for a short time (minutes) is sufficient to trigger a sustained generation of MCP-1, the major chemotactic factor for monocytes derived from endothelium, via a p38-mediated signal transduction pathway. This might be an important mechanism how platelets might change the chemotactic behaviour of the vessel wall in vivo and contribute to an early processes of atherogenesis.

4.1 Platelet–endothelium interaction and atherogenesis
There is growing evidence that endothelial dysfunction rather than denudation in combination with invasion of monocytes into the vessel wall is the major contributor to early forms of atherosclerosis. However, the pathophysiological mechanisms leading to endothelial dysfunction and the initial trigger that causes monocytes to penetrate to the subendothelium of a developing lesion are poorly understood. The hypothesis of the present work is that transient interaction of activated platelets with endothelium, which has recently been described in vivo [25,26] alters the chemotaxis and adhesive properties of endothelial cells and favors monocyte transmigration, thus initiation of atherosclerosis.

Platelets can adhere to dysfunctional endothelium in vitro [1,2] and in vivo [3,25,26]. When activated, platelets release their granule contents, which includes chemokines and growth factors that are potent triggers for inflammatory responses in endothelial cells [4]. We demonstrate that transient interactions of activated platelets for 10–60 min with HUVECs is sufficient to trigger transcription and synthesis of MCP-1, the major chemotactic factor for monocytes [27]. It has been shown in vivo that platelets can adhere intermittently to intact endothelium via several mechanisms, including the adhesion molecule P-selectin [3,25] and by binding to fibrinogen immobilized to the surface of endothelial cells via glycoprotein IIb–IIIa [26]. Thus, it seems likely that activated platelets come transiently into close contact with intact endothelium and release high concentrations of granula-stored cytokines into their thrombotic microenvironment and induce substantial MCP-1 secretion by endothelial cells. Localized platelet-induced endothelial secretion of MCP-1 and subsequent accumulation and transmigration of monocytes might be an important trigger of atherogenetic responses within the vessel wall. We found in the present study that transient interaction of activated platelets results in enhanced MCP-1 secretion and chemotaxis of monocytes towards platelet-activated HUVECs as well as enhanced monocyte–endothelium adhesion (not shown). These findings support our hypothesis that transient accumulation of activated platelets at the vessel wall (e.g. at sites of altered shear forces such as the carotid bifurcation) are predilective sites for transmigration of circulating monocytes through the endothelial surface and subsequent transformation into macrophages and foam cells (Fig. 10).

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 10 Transient interaction of activated platelets induces monocyte chemotaxis to endothelial cells.

 
4.2 Platelet-induced endothelial MCP-1 secretion is mediated via activation of the p38 MAP kinase pathway
MAPKs are key signal molecules in intracellular pathways that regulate gene expression of chemokines in activated endothelial cells. We have shown that the p38 inhibitor SB 203580 inhibits platelet-induced secretion of MCP-1. Moreover, we found that activated platelets induce activation of p38 and nuclear translocation of p38. Recently, Goebeler et al. reported that regulation of TNF-induced MCP-1 expression in HUVECs by the p38 pathway mainly occurs at the transcriptional level [14]. We found that transient interaction of platelets induce activation of the p38 kinase and MCP-1 promotor activity that can be substantially reduced using the p38 inhibitor SB 203580. Platelet-induced MCP-1 transcription and synthesis were inhibited by approximately 50% of control levels, indicating that almost half of the platelet signal leading to induction of MCP-1 expression is transmitted by the p38 cascade. This is in accordance with other reports showing a similar degree of inhibition of TNF- induced MCP-1 expression or LPS-induced IL-1 expression in monocytes [14,18]. Our data show that additional pathways which account for full platelet-induced expression of the MCP-1 gene are different from the MAP kinase signaling pathways, namely the ERK and the JNK/SAPK pathways. While basal promotor activity is dependent on a proximal promotor region containing the SP-1 site, cytokine-inducible promotor activity is mainly mediated by a distal enhancer element that contains two binding sites [28,29] and a more proximally located NF-B site [11]. Recently, we showed that activated platelets induce activation of the transcription factor NF-B [6,8]. At present we do not know how activation of the p38 system interrelates with the NF-B system in endothelial cells although the p38 cascade has been reported to be involved in NF-B-dependent gene expression in several other systems [13,18,29]. Wesselborg et al. [13] showed that inhibition of p38 by SB203580 or the dominant-negative mutant of MAPK kinase-6 (an activator of the p38 pathway), interfered with the NF-B-dependent gene expression but not with its DNA binding activity.

4.3 Limitations of the study
The present study does not provide direct evidence of whether the observed platelet-induced endothelial injury occurs in vivo. Systemic platelet activation has been described in association with cardiovascular risk factors such as hypercholesterolemia or diabetes. In these patients, an enhanced level of platelet activation might allow, mainly at the site of altered shear rates such as arterial branches, transient platelet–endothelium interaction and enhanced inflammatory responses in the endothelium. It has been shown recently that LDL sensitizes platelets to stimulation with collagen and thrombin [30]. Thus, LDL might induce a state of hypersensitivity in the platelets that could contribute to the platelet-induced endothelial secretion of MCP-1.

4.4 Therapeutic implications
A contribution of platelets to endothelial injury, monocyte adhesion and extravasation during the early stages of atherogenesis prior to any documented morphological changes of the vessel wall has been shown in in vitro studies and has been postulated for the in vivo situation. Monocytes are known to roll on and adhere to predeposited platelets [31] and circulating activated platelets assist monocyte–endothelial cell interaction under shear stress [32]. Secretion of platelet-derived compounds during platelet–vessel wall interaction might be one mechanism of accelerated atherosclerosis in heart transplant patients that reveal an enhanced level of systemic platelet activation [33] or poststenotic neointimal proliferation [34]. None of the currently available antiplatelet agents including aspirin, thienopyridines (ticlopidine, clopidogrel) or GPIIb–IIIa inhibitors alone or in combination are able to substantially reduce the release reaction and degranulation of thrombin-activated platelets [35]. Thus, targeting mechanisms of platelet secretion or platelet–endothelial interaction might give an additional option in treatment of vascular sites at high risk for atherosclerotic progression or complications (e.g. unstable vulnerable coronary plaques). For example, the endothelial cell ecto-ADPase CD39 has been shown to contribute significantly to the regulation and inhibition of platelet function [36]. As demonstrated in CD39-deficient mice interfering with CD39 mige a promising approach to modulate platelet-induced endothelial alterations [37].

Time for primary review 34 days.

	Acknowledgements

 
This study was supported by grants from the Deutsche Forschungsgemeinschaft (Ga 381/4-1, Br1026/3-3). The authors appreciate the excellent technical assistance of Antje Wallmuth.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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