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(Circulation. 1998;98:1164-1171.)
© 1998 American Heart Association, Inc.

Clinical Investigation and Reports

Activated Platelets Induce Monocyte Chemotactic Protein-1 Secretion and Surface Expression of Intercellular Adhesion Molecule-1 on Endothelial Cells

Meinrad Gawaz, MD; Franz-Josef Neumann, MD; Timm Dickfeld, MD; Werner Koch, PhD; Karl-Ludwig Laugwitz, MD; Helmut Adelsberger, PhD; Kirsten Langenbrink, BSc; Sharon Page, PhD; Dieter Neumeier, MD; Albert Schömig, MD; ; Korbinian Brand, MD

From the 1. Medizinische Klinik und Deutsches Herzzentrum and Institut für klinische Chemie und Pathobiologie (S.P., D.N., K.B.), Klinikum rechts der Isar der Technischen Universität München, Germany.

Correspondence to Meinrad Gawaz, MD, 1. Medizinische Klinikum rechts der Isar und Deutsches Herzzentrum, Technische Universität München, Lazarettstraße 36, 80636 München, Germany. E-mail gawaz{at}dhm.mhn.de

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—Platelet/endothelium interaction plays an important role in the pathophysiology of inflammation and atherosclerosis. The role of platelets for monocyte chemotactic protein-1 (MCP-1) secretion and surface expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells has been assessed.

Methods and Results—Monolayers of human umbilical vein endothelial cells were incubated with nonstimulated or ADP-activated platelets for 6 hours, and secretion of MCP-1 and surface expression of ICAM-1 were determined by ELISA and flow cytometry, respectively. In the presence of ADP-activated platelets, both MCP-1 secretion and ICAM-1 surface expression were significantly increased compared with nonstimulated platelets (P<0.02). Activation of the transcription factor nuclear factor-B (NF-B) determined by electrophoretic mobility shift assay and B-dependent transcriptional activity was enhanced in the presence of activated platelets. In addition, ADP-activated platelets induced MCP-1 and ICAM-1 promoter–dependent transcription. Liposomal transfection of a double-stranded B phosphorothioate oligonucleotide, but not of the mutated form, inhibited MCP-1 secretion and surface expression of ICAM-1 on activated endothelium (P<0.05).

Conclusions—The present study indicates that activated platelets modulate chemotactic (MCP-1) and adhesive (ICAM-1) properties of endothelial cells via an NF-B–dependent mechanism. Platelet-induced activation of the NF-B system might contribute to early inflammatory events in atherogenesis.

Key Words: platelets • endothelium • proteins • cell adhesion molecules • genes

	Introduction

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Platelet/endothelium interaction plays a central role in hemostatic and inflammatory mechanisms within the vessel wall.^{1 2 3} Dysregulation of platelet/endothelium interaction might contribute to the pathophysiology of a variety of arterial vascular disorders, including inflammation, atherosclerosis, and restenosis.^{3 4 5 6} Several lines of evidence indicate that platelet-derived substances released in close proximity to the vessel wall induce a variety of genes.⁶

Monocyte chemotactic protein-1 (MCP-1) seems to be the major chemotactic molecule generated within the vessel wall.^{7 8} MCP-1 is chemotactic for monocytes but not for neutrophils and is found in macrophage-rich areas of atherosclerotic lesions.^{6 7 8} Next to smooth muscle cells and macrophages, endothelium represents the major source of MCP-1 in the vessel wall.^{9 10} Intercellular adhesion molecule-1 (ICAM-1) is a major adhesion receptor expressed on the endothelium and is involved in monocyte adhesion to endothelial cells.^{11 12 13 14 15}

Gene expression of MCP-1 and ICAM-1 is regulated by transcription factors of the nuclear factor-B (NF-B)/Rel family.^{16 17 18} Activation of NF-B in vascular cells can be induced by a variety of signals, including the inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF), lipopolysaccharide, and oxidative and mechanical stress.^{16 17 18} Activation of NF-B has been shown to play a role in atherosclerosis.^{19 20} Recently, platelets have been shown to induce NF-B–regulated gene products in leukocytes.^{21 22}

In the present study, we investigated the effect of activated platelets on NF-B activation and production of the NF-B– regulated genes MCP-1 and ICAM-1 in endothelial cells. In addition, we assessed the feasibility and efficacy of "decoy"-B oligonucleotides transfected into endothelial cells to modulate NF-B–regulated gene expression.

	Methods

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Reagents, Oligonucleotides, and Plasmids
Single-stranded oligonucleotides were purchased as phosphorothioate diester from Perkin-Elmer. For fluorescence microscopy, oligonucleotides were used as fluorescein (FITC) conjugate. Recombinant human (rh) IL-1ß was purchased from Biermann. Monoclonal antibody anti-CD54 (ICAM-1) (clone 84H10) was from Immunotech. Apyrase, ADP, acetylsalicylic acid, and prostaglandin E₁ were from Sigma. All other reagents were of the highest purity available.

The luciferase reporter plasmids (pGL2-neo: pGL2-basic, Promega) containing 559 bp of the human MCP-1 promoter region (pGL2 neo-MCP-1) or 1297 bp of the ICAM-1 promoter region (pGL2 neo-ICAM-1), respectively, were kindly provided by Dr Nigel Mackman, Scripps Research Institute, La Jolla, Calif. Construction of the luciferase reporter plasmid pGL3 neo-MCP-1-B containing, as a tetramer, the second B element (5'-GGGAATTTCC-3') of the upstream regulatory region of the human MCP-1 gene as enhancer²³ was performed as follows. Complementary 17-mer, 5'-phosphorylated oligonucleotides (Perkin-Elmer) representing the MCP-1 gene B element were annealed and ligated into tandem repeats. The ligated B elements were inserted into the Nhel site within the polylinker of the luciferase reporter plasmid pGL3-promoter (Promega) containing the SV40 promoter but no enhancer element. A recombinant clone (pGL3 neo-MCP-1-B) containing the tetrameric B insertion in an orientation opposite to that existing in the MCP-1 gene was selected for cell culture transfections. Mutant oligonucleotides containing 3 consecutive nucleotide changes in the MCP-1 B element that prevented binding of NF-B²³ were used to construct plasmid pGL3 neo-MCP-1-mut-B serving as control plasmid.

Cell Culture
Primary human umbilical vein endothelial cells (HUVECs) were harvested by use of collagenase (Worthington) digestion as described.^{24 25 26} Cells were pooled from 3 to 6 prepared umbilical veins and were grown in 24-well culture plates (Nunc) in complete medium composed of M199 (Sigma), 10% FCS, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 mg/L streptomycin and were used as confluent monolayers after 1 to 2 passages.

Endotoxin Assay
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 experiment were evaluated by chromogenic limulus amoebocyte lysate assay (Schulz).

Incubation of Endothelial Monolayers With Platelets
Platelets were isolated from acid-citrate-dextrose–anticoagulated whole blood as described.^{25 26} Washed platelets were resuspended in Tyrode's solution–HEPES buffer (mmol/L: HEPES 2.5, NaCl 150, NaHCO₃ 12, KCl 2.5, MgCl₂ 1, CaCl₂ 2, and D-glucose 5.5, and 1 mg/mL BSA, pH 7.4) to obtain a final platelet count of 2x10⁸/mL. In experiments with nonstimulated platelets, whole blood was immediately mixed with an antiactivation cocktail that contained 1 mmol/L aspirin, 1 mmol/L theophylline, and 10 nmol/L prostaglandin E₁. In experiments with activated platelets, ADP in a final concentration of 50 µmol/L was added to the platelet suspension isolated in the absence of antiplatelet substances. Platelet suspension (200 µL) was added to 200 µL complete medium M199 (final platelet count, 1x10⁸/mL) containing 0.5 mmol/L apyrase and was transferred to wells of a 24-well culture plate covered with confluent monolayers of endothelial cells. Incubation was performed at 37°C without agitation in culture condition atmosphere for 6 hours. In some experiments, endothelial cells were incubated with membranes (1 mg/mL) isolated from nonstimulated or ADP-activated platelets as described.²⁷ Platelet releasate was obtained by removal of the supernatant after centrifugation of suspensions of nonstimulated or ADP-activated platelets (2x10⁸/mL).

Determination of MCP-1 Secretion and Surface Expression of ICAM-1
The supernatant of cultured endothelial cells treated with platelets or agonists for 6 hours was aspirated, centrifuged at 4000 rpm for 10 minutes, and stored at -80°C. Concentrations of MCP-1 protein were determined by ELISA (Biermann) with a detection limit of 5 pg/mL. Surface expression of ICAM-1 was determined by FITC-conjugated anti-CD54 monoclonal antibody and flow cytometry as described.²⁸ After aspiration of the supernatant, endothelial monolayers were incubated with anti-CD54 (50 µg/mL) and the DNA-staining fluorochrome LDS 751 (Styry 18, Exciton Inc) for 20 minutes. Thereafter, cells were mechanically detached through repetitive pipetting, and single-cell suspension was evaluated by flow cytometry for ICAM-1 immunofluorescence in the forward scatter versus LDS-fluorescence scatter plot.

Electrophoretic Mobility Shift Assay
Nuclear extracts from 1x10⁶ cells were prepared and analyzed as described.^{19 29} The B oligonucleotide (5'-AGAGTGGGAATTT-CCACTCA-3') derived from the promoter region of the human MCP-1 gene was used as a probe²⁹ and labeled by annealing of complementary primers followed by primer extension with the Klenow fragment of DNA polymerase I (Boehringer Mannheim) in the presence of [-³²P]dCTP (>3000 Ci/mmol; DuPont) and deoxynucleoside triphosphates (Boehringer Mannheim). Nuclear extracts (5 µg protein) were incubated with radiolabeled DNA probes (10 ng; 10⁵ cpm) for 30 minutes at room temperature in 20 µL of binding buffer [20 mmol/L Tris-HCl, pH 7.9, 50 mmol/L KCl, 1 mmol/L dithiothreitol, 0.5 mmol/L EDTA, 5% glycerol, 1 mg/mL BSA, 0.2% NP-40, and 50 ng of poly(dI-dC)/µL]. Samples were run in 0.25xTBE buffer (10xTBE=890 mmol/L Tris, 890 mmol/L boric acid, and 20 mmol/L EDTA, pH 8.0) on nondenaturing 4% polyacrylamide gels at 125 V. To control the nuclear protein content, the nuclear extracts were incubated with a blunt-end double-stranded Sp-1 oligonucleotide labeled with [-³²P]ATP (>5000 Ci/mmol; DuPont) and T4 polynucleotide kinase (Boehringer Mannheim). Gels were dried and analyzed by autoradiography.

Liposomal Transfection of Double-Stranded Oligonucleotides
The oligonucleotide sequence 5'-AGAGTGGGAATTTCCACTCA-3' was derived from the second B site within the human MCP-1 promoter region, which is responsible for the enhancer activity induced by IL-1ß.²³ The mutated form 5'-AGAGTGGTCCTTTCCACTCA-3' served as control. Equimolar amounts of complementary oligonucleotide strands were annealed in 0.5 mol/L NaCl for 90 minutes at 80°C. Complete annealing was verified by agarose gel electrophoresis. Endothelial monolayers were treated with a liposome (1:100 lipofectamin, Gibco)/oligonucleotide (100 nmol/L)/complete medium M199 mixture in a total volume of 500 µL per 24-well for 3 hours.

Cotransfection of Reporter Plasmids and Luciferase Assay
Twenty-four hours before transfection, endothelial cells were split and seeded in 6-well plates (50 000 cells/plate). The reporter plasmid (pGL3 neo-MCP-1-B, pGL3 neo-MCP-1-mut-B, pGL2 neo-ICAM-1, or pGL2 neo-MCP-1) was transiently cotransfected with Renilla-luciferase control plasmid pRL-TK (Promega) by incubation of HUVEC monolayers with liposomes (1:100 lipofectamin) at a plasmid concentration of 1 µg/mL for 3 hours. The firefly luciferase activity reflects gene expression under control of B (pGL3 neo-MCP-1-B), and the Renilla luciferase activity of the cotransfected reporter provides an internal control by which each value within the experimental set is normalized. After the endothelial monolayer was washed with complete medium M199, cells were incubated with platelets or agonists as indicated for 12 hours. After aspiration of the supernatant, cells were lysed by addition of reporter lysis buffer (Promega). Firefly and Renilla luciferase activity was determined with the dual-luciferase reporter assay system (Promega) with the help of a luminometer (LB953 Berthold).

Statistical Analysis
Differences between groups were tested by Student's t test for unpaired values. When the Kolmogorov-Smirnov test showed that the data were not normally distributed, we chose the Mann-Whitney-White U test for comparison of 2 groups. A value of P<0.05 was considered statistically significant. Results are presented as mean±SD.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Activated Platelets Induce MCP-1 Secretion and Surface Expression of ICAM-1 on Endothelial Cells
To evaluate the effect of platelets on secretion of MCP-1 and surface expression of ICAM-1, HUVECs were incubated for 6 hours with nonstimulated (pretreated with PGE₁, theophylline, aspirin) or ADP 50 µmol/L–activated platelets in the presence of apyrase (0.5 mmol/L). Activated platelets significantly increased secretion of MCP-1 (40% of maximal rhIL-1ß 100 pg/mL–induced secretion) (Figure 1A). Secretion of MCP-1 was dependent on activation of platelets, because significant lower MCP-1 values were found in the presence of nonstimulated platelets (P<0.02) (Figure 1A). Similarly, secretion of MCP-1 was enhanced in the presence of supernatant of ADP-activated platelets compared with supernatant derived from nonstimulated platelets (Figure 1A) (P<0.01). No significant change in MCP-1 secretion was found in the presence of membranes isolated from donor ADP-activated platelets (Figure 1A). The platelet agonist ADP 50 µmol/L in the absence of platelets did not induce significant MCP-1 secretion (Figure 1A).

View larger version (19K): [in this window] [in a new window]   Figure 1. Activated platelets induce secretion of MCP-1 and surface expression of ICAM-1 on cultured endothelium. Plots show effect of nonstimulated (1 mmol/L theophylline, 1 mmol/L acetylsalicylic acid, 10 nmol/L prostaglandine E1) and ADP 50 µmol/L–stimulated platelets, membranes, and releasate incubated with HUVECs for 6 hours on secretion of MCP-1 (A) and surface expression of ICAM-1 (B). Depicted are individual data (, , , , ), means, and SDs of 5 independent experiments.

Similarly, ADP-activated platelets induced surface expression of ICAM-1 on HUVECs (Figure 1B). Surface expression of ICAM-1 in the presence of ADP-activated platelets was significantly enhanced compared with experiments with nonstimulated platelets (P<0.05) and reached 50% of maximal inducible ICAM-1 surface expression (100 pg/mL of rhIL-1ß) (Figure 1B). Again, supernatant derived from ADP-activated platelets stimulated ICAM-1 surface expression (P<0.02), whereas no effect was found for isolated membranes (Figure 1B).

Activated Platelets Induce Activation of NF-B and MCP-1 or ICAM-1 Promoter-Dependent Transcription
Gene expression of MCP-1 and ICAM-1 is regulated by transcription factor NF-B.^{16 17 18} Thus, we asked whether activated platelets induce activation of the NF-B system. HUVECs were incubated with nonstimulated or ADP-activated platelets for 1 hour, and activation of NF-B was determined in nuclear extracts by electrophoretic mobility shift assay (EMSA) and B-dependent transcriptional activity. ADP-activated platelets induced significant activation of NF-B over baseline values (Figure 2) that was markedly enhanced compared with experiments with nonstimulated platelets (Figure 2). Binding of nuclear proteins to a double-stranded Sp-1 oligonucleotide showed that protein content was equal in all tested nuclear extracts (Figure 2). In addition, ADP-activated platelets induced B-dependent transcriptional activity in pGL3 neo-MCP-1-B transiently transfected but not in pGL3 neo-MCP-1-mut-B transfected HUVECs (P<0.01) (Figure 3A). Similarly, MCP-1 or ICAM-1 promoter-dependent transcription was increased in pGL2 neo-MCP-1 or pGL2 neo-ICAM-1 transfected cells coincubated with ADP-activated platelets (P<0.01) (Figure 3B).

View larger version (67K): [in this window] [in a new window]   Figure 2. Activated platelets induce activation of NF-B in endothelial cells. Confluent monolayers of HUVECs were incubated for 1 hour with ADP 50 µmol/L–activated platelets, nonstimulated (1 mmol/L theophylline, 1 mmol/L acetylsalicylic acid, 10 nmol/L prostaglandine E1) platelets, medium, or 100 pg/mL rhIL-1ß. Thereafter, activation of NF-B was detected in nuclear extracts by EMSA. In same extracts, binding of nuclear proteins to an Sp-1 oligonucleotide was monitored.

View larger version (26K): [in this window] [in a new window]   Figure 3. Induction of luciferase activity in endothelial cells transfected with a reporter construct containing MCP-1 NF-B site. Confluent monolayers of HUVECs were cotransfected with pGL3 neo-MCP-1-B/pRL-TK or pGL3 neo-MCP-1-mut-B/pRL-TK (A) and with pGL2 neo-MCP-1, pGL2 neo-ICAM-1, or pGL2 Basic, and pRL-TK, as indicated (B). Transfected cell monolayers were then incubated with medium alone, nonstimulated (1 mmol/L theophylline, 1 mmol/L acetylsalicylic acid, 10 nmol/L prostaglandin E1), or ADP 50 µmol/L–activated platelets or rhIL-1ß 100 pg/mL for 12 hours. Ratio of firefly/Renilla luciferase activity indicates specific activity of gene expression under control of promoter sequence. Results (mean±SD) of 4 independent experiments are shown.

B Oligonucleotides Inhibit MCP-1 Secretion and ICAM-1 Surface Expression on Activated Endothelium
Double-stranded B oligonucleotides have been shown to inhibit NF-B–regulated gene expression.^{30 31} Thus, we asked whether transfer of B oligonucleotides modulates endothelial MCP-1 secretion and surface expression of ICAM-1. HUVECs were incubated with MCP-1–B oligonucleotides for 3 hours, and secretion of MCP-1 or surface expression of ICAM-1 was determined after stimulation with rhIL-1ß for a further 6 hours. As control, a mutated form of MCP-1–B oligonucleotide, mut-B, was used that does not bind to the activated NF-B complex.²³

As shown through fluorescence microscopy, 30% to 40% of HUVECs were transfected with B oligonucleotides conjugated with FITC (Figure 4). B oligonucleotides but not the mutated form accumulated in the nucleus of rhIL-1ß–activated endothelial cells (Figure 4). In nuclear extracts of rhIL-1ß-activated and B oligonucleotide–transfected HUVECs, NF-B binding activity was significantly reduced (Figure 5). No effect on NF-B binding activity was found in HUVECs transfected with mut-B oligonucleotides (Figure 5). The binding of nuclear proteins to an oligonucleotide comprising the Sp-1 consensus sequence was not affected in cells transfected with B oligonucleotides (Figure 5).

View larger version (122K): [in this window] [in a new window]   Figure 4. Activation-dependent transfer of B oligonucleotides into nucleus of endothelial cells. Photomicrographs show uptake of fluorescein (FITC)-labeled B oligonucleotides into nucleus of cultured HUVECs. Confluent monolayers of HUVECs were incubated with lipofectamin+100 nmol/L double-stranded FITC-B oligonucleotides for 3 hours. rhIL-1ß 100 pg/mL was then added to cells for 2 hours. Cell monolayers were evaluated by laser scanning fluorescence microscopy. Evaluation of fluorescence micrographs shows that in a significant number of cells (30% to 40%), oligonucleotides accumulated in nucleus on IL-1-stimulation. Fluorescence micrographs of nonstimulated (B) and IL-1ß–activated (A) cell monolayers. Phase contrast and corresponding fluorescence micrographs of IL-1ß–activated (C) and nonstimulated (D) endothelial cells. Note that B oligonucleotides accumulate exclusively in nucleus of stimulated cells.

View larger version (48K): [in this window] [in a new window]   Figure 5. Inhibition of NF-B activation by B oligonucleotides. Confluent monolayers of HUVECs were incubated with MCP-1-B or MCP-1-mut-B oligonucleotides and rhIL-1ß as described in Methods. Activation of NF-B was evaluated in nuclear extracts by EMSA. As control, binding of nuclear proteins to oligonucleotide containing Sp-1 consensus sequence was also examined in identical nuclear extracts.

In the presence of rhIL-1ß 100 pg/mL, secretion of MCP-1 and ICAM-1 expression was significantly reduced, by 30% to 40%, in B- compared with mut-B–transfected cells (P<0.05) (Figure 6). Liposomal transfection of 100 nmol/L B oligonucleotides did not change basal secretion of MCP-1 or ICAM-1 surface expression (Figure 6).

View larger version (18K): [in this window] [in a new window]   Figure 6. Effect of B oligonucleotides on secretion of MCP-1 and surface expression of ICAM-1 on endothelial cells. Confluent monolayers of HUVECs were incubated with lipofectamin+100 nmol/L double-stranded B or mut-B oligonucleotides for 3 hours. Thereafter, medium M199, 100 pg/mL rhIL-1ß, nonstimulated, or ADP 50 µmol/L–activated platelets were added for 6 hours to cells as indicated. Secretion of MCP-1 into supernatant was determined by ELISA (A), and surface expression of ICAM-1 was evaluated by flow cytometry (B). Depicted are mean±SD of 4 independent experiments.

Platelet-induced secretion of MCP-1 and ICAM-1 surface expression of endothelial monolayers tended to be reduced in B oligonucleotide–transfected cells, although not to a statistically significant level (Figure 6).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The major findings of the present study are (1) that ADP-activated platelets induce secretion of MCP-1 and surface expression of ICAM-1 on cultured endothelial cells through an NF-B–regulated mechanism and (2) that transfection of B oligonucleotides results in reduced NF-B activation and decreases production of MCP-1 and ICAM-1 in activated endothelial cells.

The results indicate that activated platelets are able to change the chemotactic and adhesive properties of endothelial cells. This mechanism may be an important early pathophysiological event in atherogenesis or restenosis. Inhibition of NF-B–dependent gene induction might be a potential target in treatment of thrombosis-induced inflammatory and proliferative reactions in atherosclerotic arteries.

Platelet/Endothelium Interaction
Dysregulation of platelet/endothelium interaction has been implicated in atherogenesis and restenosis.^{1 2 3 4 5 6} On activation, platelets release a number of biologically highly active compounds from their granules that exert significant reactions within endothelial cells.^{32 33 34 35 36} Under pathophysiological conditions, platelets might adhere to the intact endothelial monolayer and might change the microenvironment of the vessel wall.^{1 3 26 28}

The present study shows that activated platelets induce endothelial secretion of MCP-1, the major chemotactic molecule for monocytes generated within the vessel wall.^{7 8} Significantly less endothelial MCP-1 secretion was shown in the presence of nonstimulated platelets, implying that activation-dependent release of platelet-derived products stimulates MCP-1 production. Similarly, surface expression of ICAM-1 on endothelial cells was markedly enhanced in the presence of ADP-activated platelets. As a counterreceptor for leukocytes, ICAM-1 present on the luminal aspect of endothelium is critical for leukocyte binding to the endothelium and for concomitant extravasation to sites of inflammation or injury within the vessel wall.^{11 12 13 14 15} Thus, platelet-induced secretion of MCP-1 and expression of ICAM-1 on endothelial cells might contribute significantly to entrapment and migration of monocytes, an early step in atherogenesis and restenosis.⁶

Role of Platelets and NF-B in Atherosclerosis
The importance of platelets in development of atherosclerosis has been well recognized in the past.⁶ Activated platelets adhere to inflamed endothelium or to subendothelial structures at the site of vascular injury.^{1 5} Pharmacological inhibition of platelet activation prevents ischemic complications in patients with coronary heart disease.³⁷ We²² and others²¹ have shown that activated platelets induce activation of transcription factor NF-B and related gene products in leukocytes. NF-B is a pleiotropic regulator of gene induction involved in immune and inflammatory responses.^{16 17 18 38} NF-B regulates a variety of genes coding for cytokines (MCP-1, TNF)^{16 17 18 38} and adhesion receptors (ICAM-1, vascular cell adhesion molecule-1, endothelial-leukocyte adhesion molecule-1)³⁸ that mediate endothelium-leukocyte adhesion.³⁹ NF-B–regulated gene products such as IL-1ß, MCP-1, TNF, and ICAM-1 have been found in tissue specimens of atherosclerotic lesions.⁶ Recently, activated NF-B was identified in smooth muscle cells, macrophages, and endothelial cells of human atherosclerotic tissue specimens,¹⁹ suggesting a pathophysiological role of NF-B in inflammatory and proliferative processes in atherosclerosis.²⁰

Our present findings that activated platelets induce (1) MCP-1 secretion and ICAM-1 surface expression, (2) MCP-1 or ICAM-1 promoter–dependent transcription, (3) NF-B activation as verified by gel-shift analysis and B-dependent transcriptional activity, and (4) reduction of MCP-1 and ICAM-1 production in HUVECs transfected with B oligonucleotides provide strong evidence that activated platelets significantly modulate NF-B–regulated inflammatory events in endothelial cells.

Recently, it was found that double-stranded "decoy" oligonucleotides containing the immunoglobulin B sequence bind activated NF-B and specifically inhibit NF-B–dependent transcription of gene products.^{30 31} In the cited studies, however, high micromolar concentrations of oligonucleotides were required to inhibit NF-B–induced gene expression.^{30 31} Relatively high concentrations of oligonucleotides (millimolar range) have been reported to exert significant nonspecific and toxic effects on transfected cells.^{40 41} Thus, we used a liposomal transfection protocol that allowed us to significantly reduce concentration of oligonucleotides in the nanomolar range to achieve effective inhibition of NF-B–dependent gene production. Microscopic analysis revealed no significant change in phenotype in B-transfected cells. Moreover, we were able to show by laser scanning fluorescence microscopy that B, but not mut-B, oligonucleotides accumulate in the nucleus exclusively of activated cells. Thus, we conclude that the liposomal transfection of B oligonucleotides described here allows specific inhibition of the NF-B system without significant cellular toxicity.

Study Limitations
The present study focuses on the effects of activated platelets on endothelial cells but does not address the nature of mediators released from activated platelets that might trigger NF-B–induced gene expression. NF-B can be activated by many diverse agents, such as cytokines.^{16 17 18} Activated platelets may generate (eg, thrombin) or release compounds from their granules³⁵ that might be potential activators of the NF-B system in endothelial cells. Platelets contain potent chemokines like RANTES, which is released from -granules and has been shown to be involved in NF-B–dependent MCP-1 production in human monocytes.²¹ Moreover, activated platelets have been shown to contain IL-1–like activity.⁴² IL-1 has been shown to be a major inducer of NF-B and alters adhesive and chemotactic properties of vascular endothelium.^{16 17 18 43}

Transfection of endothelial cells is limited by low efficiency and depends on the size of the DNA molecule.⁴¹ Although we show in our studies that liposomal transfection of low-molecular-weight B oligonucleotides into endothelial cells is feasible, successful DNA transfection in vivo doubtless requires higher transfection efficacy. By use of virosomes, such as Sendai virus–coated liposomes,⁴⁴ it may be possible to increase transfection efficacy significantly. Moreover, other elements of the promoter region, such as AP-1 or Sp-1, are involved in regulation of MCP-1 gene expression.²³ Thus, combined transfection of decoy oligonucleotides containing the sequence of various promoter regions might result in enhanced inhibition of NF-B–regulated gene expression.

Pathophysiological Considerations and Therapeutic Implications
The present study introduces a novel aspect of how platelets may contribute to early stages of atherosclerosis. Contact of the endothelial monolayer with activated platelets (eg, at the site of high shear conditions at vascular branches) might induce MCP-1, which might in turn enhance monocyte chemotaxis. Alteration of adhesive properties of endothelium through upregulated ICAM-1 expression might further support monocyte adhesion and transmigration. Thus, inhibition of platelet activation and accumulation at the vessel wall may be an effective strategy in downregulating atherosclerotic mechanisms.⁶

Activation of vascular cells and of circulating platelets at the injured vascular site has been suggested to contribute to restenosis.^{5 6 45 46} Balloon injury can induce activation of NF-B,^{47 48} and expression of early response genes, including MCP-1, may contribute, at least initially, to the intimal hyperplasia observed after balloon injury.⁶ In addition, expression of ICAM-1 in injured tissue has been suggested to mediate cellular inflammation processes in restenosis.⁴⁹ Thus, the NF-B system might be a potential pharmacological target to interfere with chemotactic and adhesive mechanisms within the vascular wall. The present study demonstrates that transfer of B oligonucleotides provides one mechanism by which activation of NF-B in vascular cells might be specifically inhibited. Recently, it was found that in vivo transfection of cis-element decoy against NF-B binding sites prevents myocardial infarction in animals.⁵⁰ It might be of utmost clinical interest to know whether local delivery of anti–NF-B compounds after coronary angioplasty can modulate restenotic mechanisms.

	Acknowledgments

 
This study was supported in part by grants from the Deutsche Forschungsgemeinschaft (Ga 381/2–1 to Dr Gawaz and Br 1026/3–1 and SFB469 to Dr Brand). The authors appreciate the excellent technical assistance of Caroline Bogner and Tamara Eisele.

Received April 13, 1998; accepted May 20, 1998.

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