|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2725-2734
By
From the Center for Molecular and Vascular Biology, Katholieke
Universiteit Leuven, Leuven, Belgium; and the Laboratoire de Biologie
Cellulaire, Universite Catholique de Louvain, Louvain-la-Neuve,
Belgium.
Circulating complexes of leukocytes and activated platelets are
markers for atherosclerosis, but their interaction with the arterial
endothelial lining has not been studied. Therefore, the effect of
activated platelets on rolling and adhesion of labeled human THP-1
monocytoid cells to human umbilical vein endothelial cell (HUVEC)
monolayers was studied by epifluorescence microscopy in a parallel
plate flow chamber. In the absence of activated platelets, THP-1
rolling on resting HUVEC was negligible at shear rates greater than 300 s
PLATELETS HAVE BEEN implicated in the
early development and the progression of atherosclerosis. After plaque
rupture, platelets adhere to the prothrombotic material of the plaque
interior and set off the cascade of thrombus formation and vessel
occlusion.1-3 A contribution of platelets to endothelial
injury and leukocyte extravasation into the subendothelial space during
the early stages of atherogenesis before any morphological changes of
the vessel wall can be observed has been postulated, but the mechanism
of the phenomenon remains to be defined.4,5
Activated platelets mediate leukocyte/endothelial cell interactions,
such as lymphocyte rolling on high endothelial venules of
lymphnodes.6 Platelets roll on P-selectin expressed by
endothelial cells in mesenteric venules in vivo.7 Platelets
adherent to subendothelial matrix components support rolling, adhesion,
and transmigration of leukocytes.8,9 Furthermore, platelets
deposited on subendothelial matrix exposed between shear stress
strained human umbilical vein endothelial cells (HUVEC) have been
reported to mediate leukocyte delivery to the endothelial
cells.10
Circulating activated platelets and leukocytes have been observed in
animal models and in patient populations at risk for atherosclerosis.11-14 Increased levels of low-density
lipoprotein (LDL) cholesterol induce platelet activation, as assessed
by expression of P-selectin.15 Expression of adhesion
molecules on endothelial cells is increased in conditions predisposing
to atherosclerosis.16,17 Circulating complexes of activated
platelets and leukocytes have been observed in unstable angina
pectoris18 and after mechanical revascularization of
stenosed coronary arteries,19,20 suggesting that they may
be markers of vessel wall disease and thrombogenic atherosclerotic lesions.
Injection of chemically oxidized LDL into hamsters induced complex
formation between platelets and granulocytes and increased leukocyte
rolling on microvascular endothelium exposed in a skinfold chamber.21 Thus, circulating activated platelets may
deliver leukocytes to the vessel wall under low shear conditions. The functional consequences of circulating leukocyte/platelet complexes, their adhesive properties, and the adhesion molecules involved in their
delivery to the arterial vessel wall have not yet been elucidated.
The present study evaluates whether circulating activated platelets
form complexes with monocytes and thereby enable the adhesion of
monocytes to endothelial cells under shear conditions present in the
arterial circulation. A videomicroscopic in vitro system was used,
employing cosuperfusion over resting or activated HUVEC of activated
human platelets and the human monocytoid cell line THP-1.
Reagents and materials.
Cell culture reagents, medium 199, RPMI 1640, Hank's balanced salt
solution (HBSS), phosphate-buffered saline (PBS), trypsin/EDTA, fetal
bovine serum and penicillin/streptomycin (10,000/10,000 U/mL) were
purchased from GIBCO, Lifetechnologies Inc (Paisley, UK). Human serum
was from Biowhittaker (Walkersville, MD). Paraformaldehyde and
microscopic glass coverslips 24 × 50 mm from VEL (Haasrode, Belgium) were sterilized in 70% propanol and washed in PBS. Tissue culture dishes were from Becton Dickinson Labware (Maylan Cedex, France) and uncoated Petri dishes were from Sterilin (Suffordshire, UK). For flow chamber experiments, rinsing of HUVEC monolayers and
preparation of the cell suspensions, 1% bovine serum albumin (Boehringer Mannheim, Mannheim, Germany), 200 µg/mL purified human fibrinogen, 2 mmol/L CaCl2, and 2 mmol/L MgCl2
(Sigma, St Louis, MO) were added to HBSS. This buffer is referred to as
binding buffer throughout the text. Tissue culture grade calf skin
collagen I was purchased from Boehringer Mannheim. Neuraminidase
(sialidase at 1 U/100 µL) was from Boehringer Mannheim or Sigma. For
the preparation of pH 6.5 ACD, Na3-citrate (75 mmol/L) and
dextrose (100 mmol/L) were purchased from VEL. Citric acid (38 mmol/L) was obtained from Sigma. Phorbol myristate acetate
(PMA) was dissolved in dimethyl sulfoxide
(DMSO), stored at Monoclonal antibodies.
The L-selectin blocking22 monoclonal antibody DREG56 (mouse
IgG1; Endogen, Woburn, MA) was used at 2.5 µg/mL. The monoclonal anti-P-selectin antibody CLB-Thromb/623 (mouse IgG1;
Immunotech, Marseille, France) was used at concentrations ranging from
0 to 2.5 µg/mL. For fluorescence-activated cell sorting
(FACS) analysis of P-selectin expression, the same
antibody was purchased as fluorescein isothiocyanate (FITC) conjugate.
The anti-CD34 control antibody, Birma-K324 (mouse IgG1;
Dako, Glostrup, Denmark) was used at concentrations corresponding to
those of the blocking antibodies. The anti-GP IIb/IIIa antibody
MA-16N7C2 was raised and characterized in our laboratory as described
elsewhere.25 The GPIb blocking antibody G19H10F6 was raised
against the soluble GPIb domain glycocalicin by immunization of mice
according to standard techniques.26,27 This antibody
specifically binds to GPIb in enzyme-linked immunosorbent assay
(ELISA) and blocks ristocetin- and botrocetin-induced
platelet aggregation. von Willebrand factor-dependent platelet
deposition on coated collagen at 1,300 s Culture of HUVEC and THP-1.
HUVEC were obtained by collagenase treatment28 and expanded
in Medium 199 supplemented with 10% fetal bovine serum, 10% human
serum, 100 U penicillin, and 100 U streptomycin on gelatin-coated dishes and frozen in liquid nitrogen after 2 passages. HUVEC were grown
to confluency in tissue culture flasks coated with calf skin collagen
I, trypsinized, and homogeneously seeded at high density on
collagen-coated glass coverslips placed in noncoated Petri dishes.
HUVEC were used at passage 3 to 5 for perfusion experiments. Cells were
allowed to adhere for 2 hours before media was added and cells were
grown to confluency on the coverslip. Whenever indicated, HUVEC were
activated 4 hours before the experiment by the addition of 100 nmol/L
PMA to the media. Coverslips were then carefully rinsed with binding
buffer and mounted in the flow chamber. Confluency of each coverslip
was confirmed microscopically before and after perfusion.
Platelet isolation.
Blood was drawn from healthy volunteers by puncture of an antecubital
vein with an 18G needle and freely drained on 0.1 vol citrate (108 mmol/L). Whole blood was centrifuged at 150g for 10 minutes to
obtain platelet-rich plasma (PRP), which was diluted in
1:1 vol ACD and centrifuged at 600g for 10 minutes. The
resulting pellet was resuspended in HBSS and 0.3 vol ACD was added
before the final wash at 600g for 10 minutes. Platelets were
counted and resuspended in HBSS after removing residual ACD at
300,000/µL and stored at room temperature until use within 3 hours.
An aliquot of platelets was diluted to 50,000/µL in binding buffer
and activated by the addition of 100 µmol/L TRAP for 10 minutes and
then immediatelely added to the THP-1 suspension for perfusion experiments.
FACS analysis of P-selectin expression.
Washed platelets (2.5 × 105/µL) from 3 different
donors were activated with 100 µmol/L TRAP for 10 minutes in the
presence of 1 µg/mL G4120. Samples of resting and activated platelets
were fixed at 4°C for 2 hours with 1% paraformaldehyde in
Tyrode's buffer. Platelets were washed and resuspended in Tyrode's
buffer and labeled for 20 minutes with the FITC-conjugated P-selectin antibody Thromb/6. Expression of P-selectin on the platelet membrane was analyzed using the FACS Calibur (Becton Dickinson) equipped with an
argon ion laser at wavelength 488 nm.
Flow chamber perfusion studies.
HUVEC on glass coverslips were washed free of media with binding buffer
and mounted on the bottom of a parallel plate flow chamber with a
chamber height of 0.2 mm.29 Defined shear rates between 100 and 700 s Platelet labeling and superfusion.
For direct visualization of platelet/endothelial cell interactions,
washed platelets were incubated with 1 µmol/L BCECF-AM for 20 minutes
at 300,000/µL in HBSS, activated with 100 µmol/L TRAP for 10 minutes, and then diluted to 10,000/µL in binding buffer. For
superfusions with reconstituted blood, platelets were resuspended at
10,000/µL in a mixture of packed red blood cells (purchased from the
Red Cross Blood Bank, Leuven, Belgium) and binding buffer to obtain a
hematocrit of 40%. In these experiments, no monocytes were present. In
separate sets of experiments, activated platelets were superfused at
600/µL in binding buffer for 5 minutes, followed by 5 minutes of
rinsing. Then, 2 × 105 THP-1 cells/mL were superfused
and adhesion was quantitated.
Perfusion studies with reconstituted blood and fibrinogen-coated
albumin microcapsules.
In these experiments, THP-1 cells were resuspended at 2 × 105/mL with or without 3:1 platelets in reconstituted
blood. Packed red blood cells were washed at 150g with binding
buffer to remove residual platelets before the hematocrit was adjusted
to 40%. Alternatively, THP-1 cells were resuspended in binding buffer containing 10,000 fibrinogen-coated albumin microcapsules per microliter before activated platelets were added. These microcapsules have been demonstrated to closely resemble platelets in size
distribution and recruitment to forming thrombi.30 To
exclude the possibility that rheological changes in the presence of
corpuscular particles in the perfusate caused the inhibitory effect on
THP-1 cell adhesion at higher platelet THP-1 ratios, we chose
this approach instead of nonactivated platelets to avoid interference
by mildly activated platelets generated during the platelet isolation,
because very low concentrations of activated platelets in relation to
THP-1 cells in our studies already affected THP-1 adhesion.
Deposition of platelets.
Human blood was drawn on 3.82% citrate and superfused for 5 minutes at
400 s Contribution of shear to formation of platelet/THP-1 complexes.
To elucidate whether complex formation between platelets and THP-1
cells would occur in suspension and whether exposure to shear forces
during the superfusion in the flow chamber would enhance this complex
formation, platelets were labeled with BCECF-AM, activated with 100 µmol/L TRAP for 10 minutes, and added in a ratio of 3:1 to 2 × 105 nonlabeled THP-1 cells/mL binding buffer. The number of
THP-1 cells bearing labeled platelets and the overall number of THP-1 in the cell suspension was assessed microscopically in mixed white and
fluorescent light in a Fuchs-Rosenthal counting chamber
(Electron Microscopy Sciences, Fort Washington, PA) before and after
passage of the flow chamber. The fraction of platelet decorated THP-1 cells deposited on HUVEC after 5 minutes of superfusion and 5 minutes
of buffer rinse was also counted. For each of these experiments, at
least 100 THP-1 cells on the monolayer were counted in mixed white and
fluorescent light and the fraction of platelet bearing THP-1 cells was
assessed. Similar experiments were performed in the presence of the
P-selectin blocking antibody Thromb/6 (2.5 µg/mL).
Sialidase digestion.
HUVEC were overlayed with sialidase at 0.1 U/mL and kept in an
incubator for 30 minutes. For platelet incubations, the enzyme was added to the platelets after activation to reach a final
concentration of 0.1 U/mL and then diluted greater than 100-fold in
THP-1 cell suspension after 30 minutes. THP-1 cells (10 × 106/mL) were incubated with 0.4 U/mL sialidase for 60 minutes and then diluted to 2 × 105/mL in binding
buffer for perfusion experiments. Cell suspensions were kept in a
37°C waterbath during the incubation period and the experiment.
Data analysis and statistical methods.
The recorded movies were analyzed off-line with NIH Image 1.61 (developed at the Regional Services Branch of the National Institute of
Mental Health, Bethesda, MD). The movies were reduced to
every fourth frame for analysis after detecting brightly labeled THP-1
cells. Movies were black-white inverted to facilitate further image
processing. These frames were overlayed, with the resulting image
depicting rolling cells as series of dark blobs. Rolling cells were
defined as cells moving more than their own diameter during the movie,
before arresting on the endothelium or leaving the endothelial cells to
return back to the speed of the flowing cells. The number of cells in
the 5 movies of 1 experiment spanning the full duration of the
superfusion was defined as rolling THP-1 cells per 50 seconds. Adhering
cells were recorded on 15 high-power fields of 0.06 mm2
each after 5 minutes of buffer rinse and were saved as a stack of
single frames on the hard drive of the attached computer. Because after
this time rolling cells were no longer observed, cells that were still
present on the endothelium were defined as adhering THP-1 cells/0.9
mm2. Data were processed in InStat 2.03, GraphPad Software
Inc (San Diego, CA). For comparison between groups, a
Kruskal-Wallis nonparametric ANOVA test, followed by a Dunn's multiple
comparison test, was performed. To assess differences in platelet
decoration of THP-1 cells, a Shear dependence of THP-1 adhesion to HUVEC.
Resting HUVEC did not support rolling and adhesion of THP-1 cells at
shear rates greater than 400 s
P-selectin expression on activated platelets.
TRAP activation of washed platelets induced a significant shift
of mean fluorescence values (9.2 ± 0.8 v 39 ± 5.8, n = 3, P < .05), indicating pronounced expression of P-selectin
on the platelet membrane.
Activated platelets augment THP-1 rolling and adhesion.
The addition of 1 to 50 TRAP-activated platelets per THP-1 cell to the
superfusions at 400 s
THP-1 rolling on deposited platelets.
When THP-1 cells were superfused at 400 s
Interaction of activated platelets with HUVEC.
Whether the platelet/THP-1 cell interactions that augmented
shear-dependent monocyte margination occurred in suspension or with
deposited platelets on the HUVEC surface was studied as follows. Platelet adhesion on HUVEC was directly determined by epifluorescence microscopy. Ten thousand TRAP-activated platelets per microliter, perfused at 400 s
Activated platelets and THP-1 cells form complexes under shear
conditions.
Most of the monocytoid cells had 1 or 2 platelets attached. THP-1 cells
decorated with more than 3 platelets were very rare. Complexes of
several THP-1 cells bridged by platelets were only rarely observed and
not entered in the analysis. After passage of the flow chamber, the
THP-1 number in the effluent was 123,000 ± 1,600/mL in the absence
of platelets and 92,000 ± 16,000 (n = 4) when 3:1 activated
platelets were present. Thus, 38% and 54% of the THP-1 cells were
trapped on the HUVEC monolayer. When labeled platelets were activated
and coperfused with nonlabeled THP-1 cells, 160 of 635 (n = 5 experiments) counted THP-1 cells entering the flow chamber were
decorated with platelets. The number of THP-1 cells that were decorated
with platelets in the effluent was significantly higher (318 of 764, n = 5, P < .001). Moreover, THP-1 cells recruited to the
monolayer also were preferentially decorated with platelets. Upon
addition of the P-selectin blocking antibody, the percentage of
platelet-decorated THP-1 cells in the pre-perfusion suspension was
reduced as was the recruitment of these complexes to the HUVEC and the
increase of THP-1 cell decoration in the effluent
(Table 2 and
Fig 6).
L-selectin and P-selectin mediate rolling of leukocyte/platelet
complexes.
The rolling of labeled THP-1 cells perfused over PMA-activated HUVEC in
the absence of activated platelets was inhibited by 64% after the
addition of 2.5 µg/mL of the L-selectin blocking antibody to the
superfused THP-1 cells. In the presence of 3 activated platelets
(600/µL) per THP-1 cell (200/µL), the inhibition of the increased
rolling by this antibody was 60%. The anti-P-selectin antibody did
not block rolling in the absence of platelets, but blocked 40% of the
rolling after platelets were added. Both antibodies together blocked
86% of the rolling observed in the presence of platelets, reducing the
number of rolling monocytes to control levels after L-selectin blockade
(P < .01, n = 4). Control antibody against CD34, matched for
isotype, did not block rolling or adhesion in the presence or absence
of platelets. The anti-GP IIb/IIIa antibody inhibited rolling
(Fig 7).
Role of GPIb and GPIIb/IIIa for firm adhesion of
platelet/THP-1-complexes.
Blockade of the GPIb pathway caused a trend (not significant) towards
reduced rolling of THP-1 cells in the presence of platelets. Firm
adhesion was significantly reduced by 40% (P < .05, n = 4). On the other hand, blocking of GPIIb/IIIa did not reduce monocyte adhesion in the presence of platelets (Fig 7).
Sialidase treatment of HUVEC and THP-1 abolishes rolling of
platelet/THP-1 complexes.
Treatment of platelets with 0.1 U/mL sialidase for 30 minutes did not
result in an inhibition of platelet-assisted monocyte adhesion. When
HUVEC or THP-1 cells were incubated with sialidase, the numbers of
rolling and adhering THP-1 cells in the presence of 3:1 activated
platelets at 400 s
The primary finding of the present study is that activated platelets
augment adhesion of monocytoid cells to cultured activated HUVEC in a
flow chamber system under moderate shear forces. Adhesion of THP-1
cells alone was prevented by shear forces resembling those found in
arterial vessels that are affected by atherosclerotic lesion
development in vivo. However, the addition of very small numbers of
activated platelets enabled interactions between THP-1 and endothelial
cells at higher shear rates as a consequence of decoration of the
monocytoid cells with platelets in suspension. Platelet-assisted THP-1
delivery to confluent endothelial layers was observed at shear rates of
up to 600 s Fibrinogen-coated albumin microcapsules were a generous gift from Dr
Marcel Levi (Academisch Medisch Centrum, Amsterdam, The Netherlands).
The authors are grateful to Erik Spaepen for excellent technical assistance.
Submitted December 14, 1998; accepted June 14, 1999.
Supported by the Interuniversitaire Attractiepolen, (P4/34). J.V. is
holder of the Dr. J. Choay Chair in Hemostasis Research. G.T. is on
temporary leave from the Department for Anesthesiology and Surgical
Intensive Care Medicine, Faculty of Medicine, University of
Münster Germany. G.T. received a postdoctoral fellowship from the
IMF, University of Münster, Germany.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Marc F. Hoylaerts, PhD, Center for
Molecular and Vascular Biology, Katholieke Universiteit Leuven, Campus
Gasthuisberg, O&N, Herestraat 49, B-3000 Leuven, Belgium; e-mail:
marc.hoylaerts{at}med.kuleuven.ac.be.
1.
van Zanten H, de Graaf S, Slootweg PJ, Heijnen HFG:
Increased platelet deposition on atherosclerotic coronary arteries.
J Clin Invest
93:615, 1994
2.
Ross R:
The pathogenesis of atherosclerosis: A perspective for the 1990s.
Nature
362:801, 1993[Medline]
[Order article via Infotrieve]
3.
Ip JH, Fuster V, Badimon L, Badimon J, Taubman MB, Chesebro JH:
Syndromes of accelerated atherosclerosis: Role of vascular injury and smooth muscle cell proliferation.
J Am Coll Cardiol
15:1667, 1990[Abstract]
4.
Holvoet P, Collen D:
Thrombosis and atherosclerosis.
Curr Opin Lipidol
8:320, 1997[Medline]
[Order article via Infotrieve]
5.
Zimmermann GA, McIntyre TM, Prescott SM:
Adhesion and signaling in vascular cell-cell interactions.
J Clin Invest
98:1699, 1996[Medline]
[Order article via Infotrieve]
6.
Diacovo TG, Puri KD, Warnock RA, Springer TA, von Andrian UH:
Platelet mediated lymphocyte delivery to high endothelial venules.
Science
273:252, 1996[Abstract]
7.
Frenette PS, Johnson RC, Hynes RO, Wagner DD:
Platelets roll on stimulated endothelium in vivo: An interaction mediated by endothelial P-selectin.
Proc Natl Acad Sci USA
92:7450, 1995
8.
Kuijper P, Gallardo Torres HI, Lammers J-WJ, Sixma JJ, Koendermann L, Zwaginga JJ:
Platelet and fibrin deposition at the damaged vessel wall: Cooperative substrates for neutrophil adhesion under flow conditions.
Blood
89:166, 1997
9.
Diacovo TG, Roth SJ, Buccola JM, Bainton DF, Springer TA:
Neutrophil rolling, arrest, and transmigration across activated, surface adherent platelets via sequential action of P-selectin and the beta 2-integrin CD 11b/CD18.
Blood
88:146, 1997
10.
Kuijper P, Gallardo Torres H, van der Linden JAM, Lammers J-WJ, Sixma JJ, Koendermann L, Zwaginga JJ:
Platelet-dependent primary hemostasis promotes selectin and integrin-mediated neutrophil adhesion to damaged endothelium under flow conditions.
Blood
87:3271, 1996
11.
Wolf A, Zalpour C, Theilmeier G, Wang BY, Ma A, Anderson B, Tsao PS, Cooke JP:
Dietary L-arginine supplementation normalizes platelet aggregation in hypercholesterolemic humans.
J Am Coll Cardiol
29:479, 1997[Abstract]
12.
Tsao PS, Theilmeier G, Singer AH, Leung LL, Cooke JP:
Increased platelet aggregation in hypercholesterolemic rabbits is reduced by dietary L-arginine.
Arterioscler Thromb Vasc Biol
14:1529, 1994
13.
Theilmeier G, Chan JR, Zalpour C, Anderson B, Wang BY, Wolf A, Tsao PS, Cooke JP:
Adhesiveness of mononuclear cells is increased in hypercholesterolemic humans and reduced by the NO precursor.
Arterioscler Thromb Vasc Biol
17:3557, 1997 |
TOP
ABSTRACT
INTRODUCTION
1. Activation of HUVEC with 100 nmol/L phorbol
myristate acetate (PMA) increased THP-1 cell adhesion at
shear rates less than 400 s
20°C, and added to media 1:1,000 for a final concentration of 100 nmol/L to activate HUVEC. 2',
7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM; Molecular Probes Europe, Leiden, The Netherlands) was
dissolved in DMSO, aliquoted, and stored at 
2 test was performed on all
cells that were counted under the different conditions, and the
individual fractions of platelet bearing THP-1 cells were then compared
by Fisher's exact test. A P value less than .05 was considered
significant. Data are reported as the mean ± standard error of the
mean (SEM).
) was only detectable at shear rates less than
400 s
) supported dynamic and firm interactions up to 600 s
) further augmented
THP-1 cell rolling (A) and adhesion (B) irrespective of absolute THP-1
cell number (n = 4, * P < .01).
