(Circulation. 2001;103:1688.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Aspirin (5 mmol/L) Inhibits Leukocyte Attack and Triggered Reactive Cell Proliferation in a 3D Human Coronary In Vitro Model
From the Department of Internal Medicine II, Division of Cardiology (R.V., R.F., M.O., S.V., R.B., W.K., V.H.), and the Department of Medical Microbiology (M.S.), University of Ulm, Germany.
Correspondence to Dr Rainer Voisard, Department of Internal Medicine II—Cardiology, University of Ulm, Robert-Koch-Straße 8, D-89081, Germany. E-mail rainer.voisard{at}medizin.uni-ulm.de
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Abstract |
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Background—Leukocyte attack (LA) and the triggered reactive proliferation of smooth muscle cells (SMCs) are key events for the development of early atherosclerosis and restenosis. In the present study, we used a 3D human coronary in vitro model of LA (3DLA model) to examine the effect of high-dose aspirin on the adhesion and chemotaxis of leukocytes and the reactive proliferative response of SMCs.
Methods and Results—For
dose-finding, the effect of aspirin (1, 2, 5, and 10 mmol/L) on the
tumor necrosis factor-
–induced upregulation of intercellular
adhesion molecule-1 was analyzed in monocultures of human coronary
endothelial cells (HCAEC) and the SMCs of the human coronary media
(HCMSMC). In cytoflow and Northern blot experiments, the expression of
intercellular adhesion molecule-1 was slightly reduced after incubation
with 5 mmol/L aspirin, and strong inhibition was found after incubation
with 10 mmol/L. In 3DLA models, HCAECs and HCMSMCs were cultured on
both sides of a porous filter. For LA, human monocytes or
CD4+ lymphocytes were seeded on the HCAEC
side of the 3DLA unit. A dose of 5 mmol/L aspirin inhibited the
adherence of monocytes or CD4+
lymphocytes by 50% (P<0.01)
and the chemotaxis of monocytes by 90%
(P<0.01). The reactive
proliferative response of cocultured HCMSMCs after LA, as measured by
the uptake of bromodeoxyuridine, was significantly reduced by 83%
after selective monocyte attack
(P<0.001) and by 42% after
selective CD4+ lymphocyte attack
(P<0.05).
Conclusions—A local concentration of 5 mmol/L aspirin should be accepted as the lowest rational concentration for the beneficial in vitro effects of high-dose aspirin to be reproduced in clinical studies.
Key Words: aspirin • leukocytes • atheroslerosis • restenosis
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Aspirin and its analogs are among the most widely used drugs worldwide. Therapeutic doses of aspirin exhibit 2 types of actions, depending on the dose of the drug.1 At low therapeutic doses, aspirin is an effective inhibitor of the cyclooxygenase pathway and, hence, prostaglandin-mediated signaling. At higher therapeutic doses, aspirin has antiinflammatory effects that are independent of the inhibition of prostaglandin synthesis.1
The infiltration of inflammatory leukocytes,
accumulation of cholesterol-laden macrophages, proliferation of smooth
muscle cells (SMC), deposition of extracellular matrix, and thrombosis
are crucial events that occur in early atherosclerosis and restenosis.
Among these, the localized accumulation of monocytes/macrophages and T
lymphocytes in the arterial intima after prior expression of adhesion
molecules2 3 and
triggered cellular and subcellular events are of outstanding
importance. Studies have demonstrated that coronary atherosclerotic
lesions express intercellular adhesion molecule-1 (ICAM-1) and vascular
cell adhesion
molecule-1.4 5
Inflammatory cytokines such as tumor necrosis factor- (TNF-)
upregulate ICAM-1, vascular cell adhesion molecule-1, and E-selectin,
as well as the production of chemokines (eg, monocyte chemoattractant
protein-1), at the level of gene transcription involving the binding of
nuclear factor-
B
(NF-B).6 7 8 9
Recently, Kopp and Sankar10
reported that aspirin inhibits the activation of NF-B through the
stabilization of inhibitor protein–B.
Three-dimensional human coronary in vitro models of leukocyte attack (3DLA models) focus on leukocyte adhesion and chemotaxis and the triggered proliferative response of cocultured SMCs. These models are a further development of the transfilter coculture model that was first described by Betz and colleagues.11 However, in contrast to the mechanical injury of the endothelium applied in the transfilter coculture technique,11 only an indirect injury is performed by leukocyte attack (LA) with human monocytes or human CD4+ lymphocytes in the 3DLA model.
Before the effect of high-dose aspirin was studied in the
3DLA model, dose finding was performed in monocultures of human
coronary endothelial cells (HCAEC) and HCMSMCs. As reported earlier by
our laboratory,12 the
surface expression of ICAM-1 in HCAECs and HCMSMCs is strongly
upregulated by TNF-. In part I of the study, the effect of aspirin
(1, 2, 5, and 10 mmol/L) on the TNF-–induced surface expression of
ICAM-1 was characterized. In part II of the study, the 3DLA model was
used to investigate the effect of high-dose aspirin (5 mmol/L) on the
adhesion and chemotaxis of monocytes and
CD4+ lymphocytes and on the reactive
proliferation of HCMSMCs.
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Methods |
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Cell Culture
HCMSMCs (Clonetics) were cultured in Smooth Muscle Cell Basal Medium (Clonetics). For the identification of SMCs, monoclonal antibodies against smooth muscle
-actin were used
(Renner). HCAECs (Clonetics) were grown in Endothelial Basal Medium
(Clonetics). For the identification of endothelial cells, antibodies
against von Willebrand factor (Dakopatts) were used. Human CD4+ lymphocytes and monocytes were isolated from the residual leukocytes of single donors using the MACS cell-isolation kit (Milteny Biotec GmbH).
Aspirin
A total of 500 mg of aspirin (Sigma) was dissolved in
70% ethanol (Merck). In all investigations, aspirin was added for a
period of 18 hours; during the last 6 hours of incubation, an
additional stimulus with TNF- (20 ng/mL, Sigma) was
administered.
Flow Cytometry
For flow cytometry analysis of the expression of
ICAM-1 in HCAECs and HCMSMCs, 5x105 cells
were seeded into petri dishes (75 cm2).
Aspirin (1, 2, 5, 10 mmol/L) was added to the cultures for a period of
18 hours. During the last 6 hours of aspirin incubation, the expression
of adhesion molecules was stimulated by adding TNF-.
After aspirin/TNF- treatment, cells were washed twice
with phosphate-buffered saline, detached by trypsin treatment, and
suspended in phosphate-buffered saline (pH 7.2) containing 1% bovine
serum albumin and 0.2% sodium azide at 4°C. Cells were resuspended
in 100 µL of a FITC-conjugated monoclonal antibody directed against
ICAM-1 (clone 84H10, Dianova Immunotech; final concentration 10
µg/mL) and incubated for 20 minutes at 4°C. A total of
5x103 cells (100% gated) were analyzed
immediately with a fluorescence-activated cell sorter (FACScan, Becton
Dickinson; Macintosh System Software 7.1.0).
RNA Extraction and Northern Blot
Analysis
For Northern blot studies of the effect of
aspirin/TNF- treatment on the expression of ICAM-1, monocultures of
HCAECs and HCMSMCs were incubated with aspirin (1, 2, 5, 10 mmol/L) for
a period of 18 hours. During the last 6 hours of aspirin incubation, an
additional stimulus with TNF- (20 ng/mL) was performed. Total RNA
(3x106 cells) was isolated with RNEasy Mini
Kit (Quiagen), and 10 µg of RNA was used in standard Northern blot
analysis with a ICAM-1 probe.
Phosphorimaging was used to detect the relative band density
of ICAM-1 mRNA in comparison with TNF-–stimulated cells. GAPDH was
used as a control. Experiments were performed in
triplicate.
3DLA Model
The central part of the 3DLA model is a polycarbonate
filter (diameter, 50 mm; thickness, 10 µm; pore size, 5 µm; Costar)
that separates a layer of endothelial cells from a layer of
SMCs.11 The use of HCAECs
and HCMSMCs adapts the model as closely as possible to the human
situation.13 HCAECs and
HCMSMCs were seeded in a density of 2.5x104
cells/cm2 and were supplied with the
appropriate culture
medium.13
At day 14, the 3DLA units were incubated with aspirin (5
mmol/L) for 18 hours. During the last 6 hours of aspirin incubation,
the models were treated with TNF- (20 ng/mL). For LA, the required
number of monocytes and CD4+ lymphocytes was
calculated in relation to the relative concentration of monocytes or
CD4+ lymphocytes in the full human blood.
For selective LA, 3x105 monocytes or
8x105 CD4+
lymphocytes were seeded on the HCAEC side of the 3DLA units. Selective
LA with monocytes and CD4+ lymphocytes was
studied for a period of 30 minutes, At 1, 2, 3, 4, 6, and 24 hours,
controls were performed without aspirin treatment and without TNF-
stimulus.
Adhesion and Chemotaxis Assays
All membrane filters were fixed with 4%
paraformaldehyde in phosphate-buffered saline (pH 7.4) for 3 hours at
room temperature; each filter was divided in 4 segments. One segment of
the filter was used to identify monocytes,
CD4+ lymphocytes, and ICAM-1 using indirect
immunofluorescence techniques. Monocytes were identified with primary
antibodies directed against CD68 (Dakopatts), and
CD4+ lymphocytes were identified with
primary antibodies against CD4 (Dianova). To identify ICAM-1, primary
antibodies against ICAM-1 were used (Dianova). TRITC-labeled antibodies
(goat anti-mouse, Dianova) were used as second antibodies. The number
of cells with positive staining against CD68 (monocytes) or CD4
(CD4+) was detected horizontally after
adhesion on the surface of HCAECs and after transmigration on the
HCMSMC side of the 3DLA units. The expression of ICAM-1 was only
analyzed in HCAECs according the criterion of positive or negative
upregulation after TNF- stimulus. For each investigation, 10
microscopic fields of 3 different filters were analyzed (magnification,
40x).
Triggered Cell Proliferation After LA
Proliferation of HCMSMCs was calculated 24 hours
after LA. Eighteen hours before fixation, 5-bromo-2'deoxyuridine (BrdU,
20 µmol/L, Serva) and 2'deoxy-cytidine (20 µmol/L, Serva) were
added to the culture medium of 3DLA units. Proliferation was
subsequently detected with monoclonal antibodies directed against BrdU
(Dakopatts). For second antibodies, biotinylated goat anti-mouse
antibodies were used.
One segment was embedded in Epon (Araldite, Serva,) and was polymerized at 60°C for 3 days. For histological examination, semithin sections were mounted on poly-L-lysine–coated cover slips. One section was stained with toluidine blue (Merck). In another section, araldite was dissolved from the sections using potassium hydroxide dissolved in methanol and propylene oxide immediately before immunological staining. HCAECs were identified by a positive reaction with polyclonal primary antibodies directed against anti–von Willebrand factor (Dakopatts). In each of these groups, 10 sections of 3 filters were analyzed completely (magnification, 40x), and the percentage of BrdU-positive HCMSMCs was calculated in relation to the total cell number.
Statistical Analysis
The Mann-Whitney rank-sum test was used to
investigate the significance of differences in the adhesion and
chemotaxis assays of monocytes and CD4+
lymphocytes and in the proliferation assays of HCMSMCs. Results are
expressed as mean±SD. Differences were considered significant at
P<0.05.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Identification of Cells
In monocultures of HCAECs, cells were identified by a positive reaction with antibodies directed against von Willebrand factor and by the typical "cobblestone" growth pattern in culture. Monocultures of HCMSMCs exhibited the "hill and valley" growth pattern and reacted positively with antibodies against smooth muscle
-actin.
Effect of Aspirin on Expression of ICAM-1: Flow
Cytometry Studies
The effects of aspirin (1, 2, 5, and 10 mmol/L)
on the TNF-–induced expression of ICAM-1 are demonstrated in
Figure 1
. After treatment with TNF-, a 5.6-fold
increase in the mean fluorescence levels of ICAM-1 expression in HCAECs
was found; mean fluorescence levels increased from 47.50 to
266.09. The incubation of HCAECs with aspirin in concentrations
of 1 and 2 mmol/L did not attenuate the expression of ICAM-1 in HCAEC;
mean fluorescence levels in these groups were 330.25 and 328.02,
respectively. After incubation of HCAECs with aspirin at concentrations
of 5 and 10 mmol/L, a 37% and 
80% decline in the expression of
ICAM-1 was seen, respectively; mean fluorescence levels were 167.40 and
53.99, respectively. As controls, HCAECs were incubated with
actinomycin (1 mg/mL). Incubation with 70% ethanol or adding aspirin
without prior TNF- stimulus exhibited no effect on the expression of
ICAM-1.
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In HCMSMCs, the baseline expression of mean fluorescence
levels was 38% more than that of HCAECs
(Figure 1
). After treatment with TNF-, a 2.5-fold increase
in the expression of ICAM-1 was found, and mean fluorescence increased
from 17.93 to 45.38. Treatment of HCMSMCs with aspirin in
concentrations of 1, 2, and 5 mmol/L only slightly reduced the
expression of ICAM-1 to 22%, 15%, and 29% of normal, which
corresponded to mean fluorescence levels of 35.26, 38.66, and 32.17,
respectively. A >70% decrease in ICAM-1 expression was observed after
treatment of HCMSMCs with aspirin in a concentration of 10 mmol/L. As
controls, HCMSMCs were incubated with actinomycin (1 mg/mL). Incubation
with 70% ethanol or adding aspirin without prior TNF- stimulus did
not affect the expression of ICAM-1.
Effect of Aspirin on ICAM-1 mRNA Levels:
Northern Blot Studies
After TNF- stimulus, the band density of mRNA ICAM-1
was increased 6.6-fold in HCAECs and 3-fold in HCMSMCs, which
corresponded to a relative band density of 100%
(Figure 2).
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In HCAECs, the TNF-–induced expression of ICAM-1 was
inhibited in a dose-dependent manner after incubation with aspirin in a
concentration of 5 and 10 mmol/L. Relative mRNA ICAM-1 band density was
reduced by 15% after incubation with 5 mmol/L aspirin (85±21.3% of
normal) and by 42% after incubation with 10 mmol/L aspirin
(58.3±7.7% of normal). No effect was found after incubation with 1 or
2 mmol/L aspirin (100±6.5% and 98±24.9% of normal,
respectively).
In HCMSMCs, a dose-dependent inhibition of the
TNF-–induced expression of ICAM-1 was detected after incubation
with 2, 5, and 10 mmol/L aspirin. Relative ICAM-1 RNA band density was
inhibited by 11% after incubation with 2 mmol/L aspirin (89±7.9%
of normal), by 23% after incubation with 5 mmol/L aspirin
(76.5±16.3% of normal), and by 37% after 10 mmol/L aspirin
(62.6±30% of normal). No inhibitory effect was found after incubation
with 1 mmol/L aspirin (96.8±0.8% of
normal).
3DLA Model
The 3DLA units with HCAECs and HCMSMCs were
successfully established
(Figure 3A). On the HCAEC side of the units, 1 to 2 layers of
cells were found; the superficial layer of these cells was composed of
HCAECs, as identified by a positive reaction with antibodies directed
against von Willebrand factor
(Figure 3D). On the HCMSMC side of the units, 3 to 5 cell
layers with the typical hill and valley growth pattern were
observed.
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Human monocytes and CD4+ lymphocytes were isolated from the residual leukocytes of single donors and identified by positive reaction with antibodies directed against CD68 and CD4+ lymphocytes, respectively. The purity of monocyte and CD4+ lymphocyte preparations was determined by flow cytometry. Monocyte preparations had a purity of 93%, and CD4+ lymphocyte preparations exhibited a purity of 89%.
3DLA Model: Effect of Aspirin on Leukocyte
Adhesion
In 3DLA units, the effect of aspirin (5
mmol/L) on TNF-–stimulated leukocyte adhesion, chemotaxis, ICAM-1
expression, and the proliferation of HCMSMC was studied. The effects of
aspirin on TNF-–stimulated leukocyte adhesion are depicted in
Figure 4A. No significant decrease of the number of attached
monocytes was found 30 minutes or 1 or 2 hours after adding aspirin.
Three hours after adding aspirin, the number of adherent monocytes was
significantly decreased by almost 40%
(P<0.001); 4 and 6 hours after
adding aspirin, a significant 50%
(P=0.001) and 66%
(P<0.001) decrease was found,
respectively. At 24 hours after adding aspirin, no effect on the
adhesion of monocytes was found.
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In comparison with the number of attached monocytes,
the number of adherent CD4+ lymphocytes was
decreased whether aspirin was added or not
(Figure 4B). At 30 minutes after adding aspirin, a 36%
decline of the number of adherent monocytes was found; however,
significant differences were not reached. At 1 and 2 hours after adding
aspirin, the number of adherent CD4+
lymphocytes was reduced by 36%
(P<0.01) and 39%
(P<0.01), respectively; 3, 4,
and 6 hours after adding aspirin, 39%, 73%, and 69% reductions,
respectively, of the number of adherent CD4+
lymphocytes were noticed
(P<0.001). By 24 hours after
adding aspirin, no inhibitory effect on the adhesion of
CD4+ lymphocytes was found.
In comparison with monocytes, the number of adherent CD4+ lymphocytes was always reduced, although the differences between monocytes and CD4+ lymphocytes were smaller with increasing time after adding aspirin. At 30 minutes and 1 and 2 hours after seeding, the number of adherent CD4+ lymphocytes had decreased by 56%, 55%, and 64%, respectively, in relation to the correspond-ing numbers of adherent monocytes. At 3, 4, and 6 hours after adding aspirin, the differences between monocytes and CD4+ lymphocytes were getting smaller and the number of adherent CD4+ lymphocytes was decreased in comparison with the corresponding number of adherent monocytes by 44%, 38%, and 24%, respectively. By 24 hours after adding aspirin, the number of adherent CD4+ lymphocytes was decreased to 19% in comparison with monocytes.
Effect of Aspirin on Leukocyte Chemotaxis in
3DLA Units
Chemotaxis on the HCMSMC side of the 3DLA
units was found in only 10% to 20% of the adherent monocytes on that
side
(Figure 5A). No chemotaxis was detected after
CD4+ lymphocyte attack
(Figure 5B). The effect of aspirin (5 mmol/L) on the
chemotaxis of leukocytes from the HCAEC side to the HCMSMC side of the
3DLA units is depicted in
Figure 5.
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TNF- stimulus increased the number of transmigrated
monocytes in comparison with the group without prior stimulus; however,
significant differences were only seen 6 hours after monocyte attack
(P<0.01). An 8-fold and 6-fold
increase of the number of monocytes was found 2 and 3 hours after
seeding, respectively, and 4 and 24 hours after seeding, the increase
was 1.8- and 4-fold, respectively
(P=NS for all).
CD4+ lymphocytes were never detected on the
HCMSMC side of the units.
Effect of Aspirin on Proliferative Activity of
HCMSMCs in 3DLA Units
Proliferation of HCMSMCs was analyzed in
TNF-–stimulated 3DLA units after selective monocyte and selective
CD4+ lymphocyte attack. The data are
presented in
Figures 3B and 6.
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In comparison with the baseline proliferation of HCMSMCs in 3DLA units (2.8±2.3 cell divisions), selective monocyte and CD4+ lymphocyte attack significantly increased proliferation (6.6±3.6 cell divisions, P<0.001, and 4.3±3.2 cell divisions, P<0.001, respectively). Proliferation of HCMSMCs after selective monocyte attack was significantly increased (P<0.001) in comparison with the proliferation of HCMSMCs after selective CD4+ lymphocyte attack.
Incubation with 5 mmol/L aspirin significantly inhibited the proliferation of HCMSMCs in 3DLA units after both selective monocyte or CD4+ lymphocyte attack. A 83% decrease (P<0.001) was detected after selective monocyte attack (1.1±1.6 cell divisions), and a 42% decrease (P<0.05) was detected after selective CD4+ lymphocyte attack (2.5±2.2 cell divisions).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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LA and triggered cellular and subcellular reactions are pivotal events of early atherosclerosis and restenosis. We came to 3 basic conclusions after completing the present study. First, in monocultures of HCAECs and HCMSMCs, the surface expression of ICAM-1 was reduced in a dose-dependent manner after incubation with 5 and 10 mmol/L aspirin, but no effect was detected after incubation with 1 and 2 mmol/L aspirin. Second, in the 3DLA model, 5 mmol/L aspirin significantly inhibited the adherence of monocytes and CD4+ lymphocytes and the chemotaxis of monocytes. Third, 5 mmol/L aspirin significantly reduced the reactive proliferative response of cocultured HCMSMCs after selective monocyte or CD4+ lymphocyte attack in the 3DLA model.
A dose-dependent inhibitory effect of aspirin on
NF-B–mediated signal cascades agrees with the prior reports of
Weber et al14 and Amberger
et al,15 who studied
endothelial cells from human umbilical veins. The exact mechanism of
aspirin on TNF-–induced and NF-B–mediated events is not
entirely clear. NF-B comprises a family of cellular transcription
factors that are involved in the inducible expression of a variety of
cellular genes that regulate the inflammatory
response.16 17
In 3DLA units, 5 mmol/L aspirin significantly inhibited
adhesion after selective monocyte or CD4+
lymphocyte attack. These data match the preceding reports of Gerli et
al18 and Amberger et
al,15 which describe an
inhibitory effect of 5 mmol/L aspirin on the adhesion of human T
lymphocytes in human umbilical vein endothelial cells. A dose of 5
mmol/L aspirin significantly inhibited the triggered reactive
proliferation of HCMSMCs after both selective monocyte and
CD4+ lymphocyte attack. Moreover, aspirin (5
mmol/L) significantly inhibited the TNF-–induced proliferation of
HCMSMCs without selective LA. These data are in conformity with a
study by Osnes and
colleagues,19 who reported a
50% inhibition of TNF- in the supernatant of human monocytes after
adding aspirin in a concentration of 5 mmol/L.
Plasma levels of aspirin in vitro and in vivo are complex to
calculate because aspirin is rapidly hydrolyzed to salicylic acid by
nonspecific esterases. Hydrolysis occurs in the liver and, to a lesser
extent, the stomach, so that only 68% of the dose reaches the systemic
circulation as aspirin.20
Recently, Cerek et al21
reported that aspirin (100
mg · kg–1 · d–1)
significantly inhibited reactive cell proliferation after angioplasty
in male rats. Data on rat plasma levels after the administration of 100
mg · kg–1 · d–1
aspirin are not available (to our knowledge); however, it has been
reported that a dosage of 200
mg · kg–1 · d–1
results in peak plasma concentrations of 5
µg/mL,22 which corresponds
to 0.03 mmol/L. In humans, the peak plasma levels of aspirin rise 20
minutes after the oral administration of 650 mg/d to a concentration of
25 µg/mL (which corresponds to 0.15
mmol/L).23 These levels
decline to <5 µg/mL after 2 hours. Plasma salicylate concentrations
raise equally rapidly, reaching a peak of 45 µg/mL by 1 hour after
administration.23
Despite these low plasma levels after oral administration,
aspirin has positive effects on carotid plaque growth (at doses of 50
mg and 900 mg per day),24
endothelial dysfunction (at 1000 mg per
day),25 and the reduction of
proinflammatory cytokines (at 300 mg per
day).26 With reference to
the fact that, in humans, peak concentrations of 0.15 mmol/L can be
obtained after the oral administration of 650 mg of
aspirin,23 it becomes
evident that orders of magnitudes exist in comparison with the
concentration of 5 mmol/L aspirin that was proven effective in the
present study. Thus, the beneficial effects of aspirin in the clinical
trials
cited24 25 26
have not been caused by the inhibitory effect of aspirin on leukocyte
adhesion and chemotaxis or the triggered proliferative response of
HCMSMCs.
In accordance with the data presented here, it was
previously demonstrated in large clinical trials that the daily
administration of 330 mg of aspirin (which corresponds to 0.1
mmol/L)27 or 1000 mg of
aspirin (which corresponds to 0.2
mmol/L)28 are not successful
to prevent restenosis after coronary peripheral angioplasty. The
discrepancy between the positive effect of a concentration of 0.03
mmol/L aspirin to inhibit restenosis in
rats21 and the negative
effect of an increased concentration in clinical
trials27 28
underlines the need for complex in vitro models that more closely mimic
the clinical situation.
The authors suggest accepting the fact that a permanent local concentration of aspirin of 5 mmol/L is the lowest rational concentration to obtain the reported beneficial in vitro effects of high-dose aspirin in clinical studies. Recently, Laham and colleagues29 opened the door to the use of the pericardium as an perivascular drug depot. Further studies are mandatory to investigate whether the perivascular administration of high-dose aspirin (5 mmol/L) opens the field for a new cardiovascular indication for one of the oldest and most widely used drugs in the world.
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Acknowledgments |
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This work was supported by Bayer-Vital GmbH, Cologne, Germany.
Received July 31, 2000; revision received September 22, 2000; accepted September 24, 2000.
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