© 1997 American Heart Association, Inc.
Articles |
Angiotensin II–Induced Leukocyte Adhesion on Human Coronary Endothelial Cells Is Mediated by E-Selectin
From the Departments of Internal Medicine/Cardiology and Angiology (M.G., W.A.-S., V.R.-Z., P.B., K.G., E.F.), Virchow Klinikum, Humboldt University and German Heart Institute, Berlin, the Department of Cardiovascular Surgery (M.L.), German Heart Institute, Berlin, and the Institute of Physiology (A.Z., P.G.), Free University Berlin (Germany).
Correspondence to Michael Gräfe, MD, Department of Internal Medicine/Cardiology, German Heart Institute, Augustenburger Platz 1, D-13353 Berlin, Germany.
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Abstract |
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Abstract Clinical data suggest a link between the activation of the renin-angiotensin system and cardiovascular ischemic events. Leukocyte accumulation in the vessel wall is a hallmark of early atherosclerosis and plaque progression. E-Selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) are adhesion molecules participating in mediating interactions between leukocytes and endothelial cells and have been found to be expressed in atherosclerotic plaques. We investigated whether angiotensin II, the effector of the renin-angiotensin system, influences the endothelial expression of E-selectin, VCAM-1, and ICAM-1. In coronary endothelial cells derived from explanted human hearts, angiotensin II (10-11 to 10-5 mol/L) induced a concentration-dependent increase in E-selectin expression. The effect was measured by cell ELISA and duplex reverse-transcription polymerase chain reaction (RT-PCR) and reached its maximum at 10-7 mol/L. Angiotensin II induced only a small increase in E-selectin expression in cardiac microvascular endothelial cells. VCAM-1 and ICAM-1 were not affected by angiotensin II stimulation. In addition, the effect of angiotensin II–induced E-selectin expression on leukocyte adhesion was quantified under flow conditions. Angiotensin II (10-7 mol/L) increased leukocyte adhesion significantly to 67% of the maximal effect by tumor necrosis factor-
at a wall shear stress of 2 dyne/cm2. This
adhesion was found to be E-selectin dependent, as demonstrated by
blocking antibodies. The AT1-receptor
antagonist DUP 753 significantly reduced
E-selectin–dependent adhesion, whereas the AT2-receptor
antagonist PD 123177 had no inhibitory effect.
In addition, only AT1-receptor, but not
AT2-receptor, mRNA could be detected by RT-PCR in
coronary endothelial cells. Therefore, it is
suggested that AT1 receptors mediate the effects of
angiotensin II on E-selectin expression and leukocyte
adhesion on coronary endothelial cells.
Key Words: endothelial cell • angiotensin II • E-selectin
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Introduction |
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Accumulation of monocytes and lymphocytes in the vessel wall is a hallmark of the early stages of atherosclerosis.1 2 The adhesion molecules E-selectin, VCAM-1, and ICAM-1 support monocyte adhesion to and interaction with endothelial cells and, hence, may play a crucial role in the pathogenesis of atherosclerosis. VCAM-1 and ICAM-1 have been identified on endothelial cells covering atherosclerotic lesions in human coronary arteries. E-Selectin, VCAM-1, and ICAM-1 mediate the adhesion of leukocytes by interaction with leukocyte VLA-4 (CD29/CD49d), LFA-1 (CD11a/CD18), and Mac-1 (CD11b/CD18), respectively.3 The expression of E-selectin that mediates the adhesion of neutrophils as well as certain T lymphocytes and monocytes is restricted to endothelial cells. E-Selectin interacts with leukocyte sialyl-Lewisx as a ligand and has been recently shown to be expressed in human atherosclerotic lesions.4
Clinical observations suggest a link between augmented renin-angiotensin system activity and the development of cardiac ischemic events.5 6 Studies with ACE inhibitors have demonstrated various cardiac and vascular protective effects,7 but the relevant mechanisms of action are not yet fully understood. In animal models of cardiovascular disease, ACE inhibitors reduce the development of atherosclerotic lesions,8 restore impaired endothelial function,9 and reduce neointimal hyperplastic responses after denudation of the endothelium.10 Specific binding sites for angiotensin II on endothelial cells have been described,11 and angiotensin II appears to have a modulating effect on several endothelial cell functions, such as prostacyclin, endothelin secretion,12 13 and endothelial cell growth.14
In view of a possible functional relationship between the renin-angiotensin system and leukocyte adhesion in the pathophysiology of coronary atherosclerosis, the present study was designed to investigate the effects of angiotensin II on the expression and modulation of E-selectin, VCAM-1, and ICAM-1 on human cardiac endothelial cells and on leukocyte adhesion. These experiments were performed with endothelial cells from epicardial coronary arteries and from microvessels of human hearts, since previous studies have shown functional differences between these cells in regard to their sensitivity to peptides and their ability to promote leukocyte adhesion.15 16
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Materials and Methods |
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HCECs
Endothelial cell cultures were prepared as described previously.17 Briefly, segments of epicardial coronary arteries were obtained immediately after cardiac explantation from patients with dilated cardiomyopathy and stored in ice-cold medium 199 (Seromed) until further preparation. Within 24 hours, the vessels were cleared of the surrounding tissue and incubated in 0.2% collagenase II (Roth) at 37°C for 30 minutes. Detached cells were flushed out by rinsing the vessels thoroughly with medium 199. The cells were pelleted and seeded on gelatin-coated T-25 flasks (Falcon). Cells from a 10-cm vessel segment were seeded on a 25-cm2 growth area. The cells were grown in medium 199 containing 20% FCS (GIBCO-BRL), antibiotics (100 µg/mL streptomycin and 100 U/mL penicillin, both from Sigma), 10 mmol/L HEPES buffer (Sigma), and 10 ng/mL endothelial cell growth factor (Boehringer-Mannheim) in 5% CO2 at 100% humidity and 37°C. The endothelial cells were separated from nonendothelial cells with paramagnetic beads linked to the Ulex europaeus I lectin. The purity of the cultures (>98% endothelial cells) was evaluated by fluorescence-activated cell sorting after labeling with DiI-Ac-LDL or CD31.
Human Cardiac Microvascular Endothelial Cells
For isolation of microvascular endothelial
cells, heart muscle segments were enzymatically digested as described
previously17 and grown under culture conditions identical
to those used for coronary endothelial cells.
Nonendothelial cells were removed from the cultures by
treatment with Ulex europaeus–linked paramagnetic beads,
resulting in a purity of the cultures comparable to that of HCECs.
Adhesion Assay
A perfusion chamber, as previously described,18
provided near-physiological flow conditions in
vitro by the use of a parallel-plate geometry and a well-defined low
Reynolds number flow over the endothelial cell
monolayer. The coverslip with the cell monolayer and the bottom plate
were separated by a gasket, which defined the height and width of the
flow channel. The flow channel was provided with a narrow inlet and
widened toward the outlet in a hyperbolic manner, thus producing a
range of linearly decreasing shear stresses in the direction of flow.
The local wall shear stress as a function of the distance from the
entrance port is given by the following equation: 
=6 ·
· Q · (1-l/L)/(h2w), where is
shear stress, is viscosity of the perfusion medium (0.75 cp), Q is
flow rate (116 µL/min), l is distance from the entrance port (2 to
30 mm), L is flow channel length (38 mm), h is channel height
(120 µm), and w is channel width at the entrance (2.2
mm).
The assembled flow chamber was placed on the stage of an inverted Nikon
microscope, and its temperature was maintained at 37°C throughout the
experiments by feedback-controlled warm air flow. An injection port at
the entrance permitted the addition of cell suspensions to the main
stream of the perfusion medium. Usually, 100 µL of HL-60 cell
suspension (106 cells/mL) was added, and cell adhesion was
allowed to take place during a 5-minute run. The number of adherent
cells, counted after 5 minutes with the perfusion medium still flowing,
represents a composite measure of attachment rate, detachment
rate, and rolling velocity. In each treatment group, the number of
stable adherent cells was determined per field of view. In all
experiments, results are presented as the number percent of
adherent cells in positive control experiments in which adhesion
to TNF-–activated endothelial cells was
measured.
HCECs of the second passage were grown on glass coverslips coated with
5 µg/cm2 fibronectin (Boehringer-Mannheim)
to facilitate tight adherence of the cells. Confluent monolayers were
stimulated either with TNF- (100 U/mL) or with
angiotensin II (10-7 mol/L)
alone or in the presence of the AT1-receptor
antagonist DUP 753 (10-5
mol/L, DuPont) or the AT2-receptor
antagonist PD 123177 (10-5
mol/L, Parke-Davis). The coverslips were mounted in the flow
chamber and superfused with 105 HL-60 cells suspended in
medium 199 containing 20 mmol/L HEPES and 0.1% BSA for 5
minutes at a flow rate of 116 µL/min. In some experiments, stimulated
endothelial monolayers were incubated with a blocking
anti–E-selectin antibody (10 µg/mL BBA2, British
Biotechnology, Biermann) for 30 minutes. An isotype-matched murine
anti-CD4 antibody served as a control antibody. Endotoxin contamination
of media and drugs was routinely checked by a limulus amebocyte lysate
Endo-LAL test (Chromgenix AB, Pharmacia LKB) and was found to be <0.06
endotoxin units/mL.
Cell ELISA
HCECs of the second passage were plated on gelatin-coated
96-well tissue culture plates. Confluent cultures were stimulated with
angiotensin II (10-11 to
10-5 mol/L) for 4 hours in culture
medium containing 20% FCS. In additional experiments, the time course
of protein expression was not found to be increased significantly
during longer stimulation periods. In each experiment, TNF- (1000
U/mL)–stimulated endothelial cells served as positive
controls; unstimulated cells served as negative controls. After
incubation, cells were rinsed with PBS and incubated with monoclonal
antibodies against E-selectin, VCAM-1, and ICAM-1 (all British
Biotechnology, Biermann) for 30 minutes at 37°C. Binding saturation
was achieved with a dilution of 1:400 for the E-selectin antibody and
1:200 for antibodies directed against VCAM-1 or ICAM-1. The cultures
were then washed twice with PBS and further incubated with an alkaline
phosphatase–labeled anti-mouse antibody (Sigma) at a dilution of
1:3000 for 30 minutes. After three washings, the binding of the
antibodies was assessed by addition of 100 µL
p-nitrophenyl phosphate (Sigma) in diethanolamine buffer
(Sigma). Absorbance was elucidated on an ELISA reader (MR 7000,
Dynatech GmbH) at 405 nm after 45 minutes.
RT-PCR Analysis
Total RNA was prepared by a method described by Chomczynski and
Sacchi19 from human coronary
endothelial cells after stimulation with
angiotensin II (10-7 mol/L)
or TNF- (1000 U/mL) for 4 hours. First-strand cDNA was synthesized
by M-MLV reverse transcriptase (GIBCO BRL) using oligo(dT) primers.
The reaction was carried out in a 20 µL final volume for 50 minutes
at 42°C. A duplex PCR was performed on a Perkin-Elmer DNA cycler
using 2 µL of the transcription mixture, 4 U of Taq polymerase
(Perkin-Elmer), 0.2 mmol/L dNTPs, and 10x reaction puffer
(100 mmol/L Tris-HCl [pH 8.3], 500 mmol/L
KCl, 15 mmol/L MgCl2, and 0.01% gelatin). Each
primer (20 pmol) was added to 25-µL reaction volumes. Two specific
20-mers each for E-selectin,20 VCAM-1,21 and
PDH22 were chosen as primers, with each primer pair
spanning at least one intron. Information on the primer sequences used
is summarized in the Table
. The duplex
PCR with primers for E-selectin or VCAM-1 and the internal standard PDH
was performed in one tube and showed a linear increase of the
amplification products up to 30 cycles. Twenty-six cycles were used
in all experiments. Amplified solution (5 µL) was run in a 3%
agarose gel electrophoresis in Tris-borate/EDTA buffer and stained with
0.5 µg/mL ethidium bromide. For analysis of AT
receptors, the isolated mRNA was subjected to additional DNase
digestion using an optimized protocol23 to remove possible
DNA contamination, since both angiotensin receptor
sequences contain no intron in the coding sequence.24 DNA
contamination was monitored by PDH amplification (mRNA, 103 bp; DNA,
185 bp) and amplification of the mRNA before reverse transcription
after 40 cycles. Each primer (16 pmol) was added, and amplification
products were analyzed after 40 cycles on a 3% agarose
gel. Human uterus served as a positive control tissue for
AT2-receptor RT-PCR. Information on the primer sequences
used is summarized in the Table.
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PCR products for duplex PCR were analyzed by HPLC as described by Katz and Haff25 on a TSK-DEAE column (3.5x4.6 mm, Perkin-Elmer). The area under the curve was measured for the PDH, VCAM-1, and E-selectin peaks. The ratio between the peak area for E-selectin or VCAM-1 and the peak area for PDH was taken as an index of the mRNA level for the adhesion molecules. Double measurements of PCR products were performed. The results reported were obtained in three separate experiments and expressed as mean±SEM.
Statistics
Statistical significance was analyzed by one-way ANOVA
and t test for unpaired observations, as appropriate.
Significance was assumed at a value of P<.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Modulation of E-Selectin, VCAM-1, and ICAM-1 Protein Expression
Stimulation of human coronary endothelial cells for 4 hours with concentrations of TNF-
between 0.1 and 2000
U/mL showed a concentration-dependent increase of all three
endothelial adhesion molecules. Half-maximal
stimulation of E-selectin was observed at 1 U/mL TNF- (Fig 1a); half-maximal stimulation of VCAM-1
and ICAM-1 was observed at 5 to 10 U/mL TNF- (Fig 1b and 1c). In
cultures of microvascular endothelial cells, TNF-
also induced an increase in E-selectin expression, but half-maximal
effects were obtained at higher concentrations of TNF- (10 to 100
U/mL, Fig 1d).
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, a),
VCAM-1 (
, b), and ICAM-1 (
, c) was measured by cell
ELISA. Effects of TNF-
Stimulation of human coronary endothelial cells
with angiotensin II in concentrations ranging from
10-11 to 10-5
mol/L caused a concentration-dependent increase in E-selectin
expression, with maximal effects at10-7
mol/L. The maximal effect of angiotensin II reached
54% of the maximal effect induced by TNF- (Fig 2a). In contrast to TNF-, stimulation
with angiotensin II did not significantly change the
expression of VCAM-1 (Fig 2b) or ICAM-1 (Fig 2c). The effect of
angiotensin II on the expression of E-selectin was lower in
microvascular endothelial cells compared with
macrovascular endothelial cells; it also reached its
maximum at 
10-7 mol/L (Fig 2d).
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Modulation of E-Selectin and VCAM-1 mRNA Content in
Coronary Endothelial Cells
Neither TNF- (1000 U/mL) nor angiotensin II
(10-7 mol/L) affected mRNA levels for
PDH and was therefore used as an internal standard. Stimulatory effects
of TNF- and angiotensin II on mRNA of E-selectin and
VCAM-1 were assessed from the ratios of E-selectin mRNA and VCAM-1 mRNA
over the mRNA of PDH. TNF- (1000 U/mL) significantly increased
E-selectin/PDH from 0.61±0.2 to 4.15±0.34 and VCAM-1/PDH from
1.01±0.2 to 7.55±1.73 (for both, P<.05).
Angiotensin II increased E-selectin/PDH to 1.6±0.2
(P<.05 versus control, Fig 3). No change in mRNA levels was detected
for VCAM-1. After stimulation of coronary
endothelial cells with angiotensin II, mRNA
of E-selectin increased 3-fold compared with baseline values and
reached 38% of the stimulatory effects of TNF-.
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Effect of Angiotensin II on Leukocyte Adhesion
In order to determine whether an angiotensin
II–induced E-selectin expression increases leukocyte adhesion to
endothelial cells, adhesion was measured in a flow
chamber adhesion assay at wall shear stresses between 0.9 and 2.6
dyne/cm2. Within this range of shear stresses, adhesion
requires selectin-mediated or 4-integrin–mediated
interactions. In general, adhesion of HL-60 cells to human
coronary endothelium decreases with increasing
wall shear stress. Stimulation with TNF- induced a significant
increase in HL-60 cell adhesion at all shear stresses. At 2
dyne/cm2, 34±9 cells adhered to TNF-–stimulated
endothelial cells, whereas at 0.9 dyne/cm2,
103±11 adherent cells were counted per field of view. All further
observations were expressed as percentage of the TNF-–induced
adhesion. Furthermore, a significant increase in HL-60 cell adhesion
was observed after stimulation with angiotensin II
(10-7 mol/L) and differed significantly
from unstimulated cells within the observed shear stress range (Fig 4a). At a shear stress of 2
dyne/cm2, the angiotensin II–induced adhesion
reached 55% of maximal TNF-–induced effects. Antibodies against
E-selectin (BBA2) blocked the TNF-–induced adhesion completely. On
angiotensin II–stimulated cells, the antibody did not
block the HL-60 adhesion completely. However, the remaining difference
did not reach statistical significance compared with control cells.
Anti-CD4 antibodies, which served as a nonbinding isotype-matched
control antibody, did not significantly reduce HL-60 cell adhesion to
angiotensin II–stimulated endothelial
cells (Fig 4b).
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) for 4
hours, and adhesion was expressed as percentage of
maximum adhesion induced by TNF-
). b, Adhesion of HL-60 cells to
coronary endothelial cells at 2.0
dyne/cm2. Cells were stimulated by angiotensin
II (10-7 mol/L) for 4 hours, and adhesion was
expressed in relation to TNF-
The effects of angiotensin II receptor
antagonists on HL-60 cell adhesion were evaluated in a
second set of experiments. However, in this series, adhesion induced by
angiotensin II was somewhat lower but significantly higher
compared with control cell adhesion within the shear stress range. At 2
dyne/cm2, the AT1-receptor
antagonist DUP 753 (10-5
mol/L) significantly reduced adhesion to 34% of the
angiotensin II–induced adhesion, whereas PD 123177
(10-5 mol/L), the
AT2-receptor antagonist, had no significant
effect on leukocyte adhesion. Both inhibitors together did
not exceed the effect of the AT1-receptor
antagonist alone (Fig 5).
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AT-Receptor mRNA in Coronary Endothelial Cells
The mRNA of both known angiotensin II receptors was
measured by RT-PCR. The presence of DNA was monitored by amplification
of mRNA before reverse transcription and PDH gene amplification, which
yielded a 103-bp band with mRNA and a 185-bp band with DNA (Fig 6a). Amplification of cDNA with primers
of the AT1 receptor resulted in a 174-bp signal, whereas no
mRNA of the AT2 receptor could be demonstrated. However,
tissue from human uterus yielded a signal for the AT2
receptor (Fig 6b).
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The present study demonstrates the modulated expression of the adhesion molecule E-selectin via angiotensin II in human coronary endothelial cells. Evidence for this pathway was obtained from measurements of increased antigen expression at the protein level as well as from increased mRNA content. Functionally, the angiotensin II–induced expression of E-selectin leads to an increase of HL-60 cell adhesion, as demonstrated under near-physiological flow conditions. The effects of angiotensin II on E-selectin expression appear to be mediated by an AT1 (angiotensin II) receptor, since the AT1-receptor antagonist DUP 753 but not the AT2-receptor antagonist PD 123177 suppressed E-selectin–dependent adhesion. These observations indicate a link between the renin-angiotensin system and the expression of E-selectin, which is thought to play a crucial role in the processes of inflammation and atherosclerosis.1 26 27
Adhesion molecule expression on endothelial cells after
stimulation with angiotensin II was quantified by cell
ELISA.28 Strong upregulation of E-selectin, VCAM-1, and
ICAM-1 was detected by cell ELISA after incubation with TNF-, which
confirms a regulation pathway of adhesion molecules by
cytokines in endothelial cells derived from
human epicardial coronary arteries. Here, stimulatory effects
on E-selectin expression were detected after stimulation with TNF-
at concentrations as low as 0.1 U/mL. Interestingly, upregulation of
E-selectin in coronary artery endothelial cells
turned out to be very sensitive compared with cardiac microvascular
endothelial cells, since maximal stimulation of
E-selectin by TNF- was obtained at 100-fold lower concentrations.
Compared with E-selectin, maximal upregulation of VCAM-1 and ICAM-1 was
observed at a 10-fold higher concentration of TNF- in
coronary artery endothelial cells. The
differential effects on E-selectin expression confirm the differences
in the sensitivity of these cells to peptides.15 This
observation may indicate that different regulatory mechanisms are
involved in leukocyte rolling and adhesion in epicardial
coronary arteries and microvessels of the heart, as is also
demonstrated by the preferential occurrence of an L-selectin ligand in
microvascular endothelial cells.16 Whether
the differences of cardiac macrovascular and microvascular
endothelial cells have an impact under
pathophysiological conditions would require further
investigation.
Current knowledge on the regulation of E-selectin, VCAM-1, and ICAM-1
by TNF- is extended by the present findings, which show that
angiotensin II also induces E-selectin expression in
endothelial cells. This is the first report showing
that activation of the renin-angiotensin system induces
the expression of E-selectin. Similar to the expression of E-selectin
after TNF- stimulation, the expression of E-selectin is less
sensitive after angiotensin II stimulation of microvascular
endothelial cells compared with macrovascular
coronary endothelial cells. The expression of
E-selectin reported here is rather selective, since
angiotensin II does not affect the expression of VCAM-1 or
ICAM-1, whereas (and in contrast) all three adhesion molecules are
stimulated by TNF-. This may indicate distinct intracellular signal
transduction pathways in the regulation of E-selectin, VCAM-1, and
ICAM-1.
The effective concentration range of angiotensin II that stimulates E-selectin expression and HL-60 cell adhesion is within the same range in which angiotensin II exerts its effects, for example, on the regulation of vascular tone in vitro.29 This suggests that angiotensin II–induced adhesion phenomena may indeed have physiological and pathological relevance.
An increase of expression of E-selectin induced by
angiotensin II at the protein level is paralleled by an
increase of E-selectin mRNA as quantified by a duplex RT-PCR
system.30 31 The target mRNA was normalized to an internal
standard. This excludes variations of RNA processing during isolation
and reverse transcription. PDH is well suited as an internal standard
for RT-PCR, since no pseudogenes of this gene have been reported to
date,32 and its mRNA-level is not affected by stimulation
with TNF- or angiotensin II. The PCR reaction in the
same tube under exactly the same conditions further ensures that
variations during the amplification process are leveled off. With this
system, we confirmed the data obtained with the adhesion assay and the
cell ELISA showing a 3-fold increase in E-selectin mRNA in response to
angiotensin II. However, no increase in mRNA was detected
for VCAM-1.
The selectin family of adhesion receptors has been implicated in
initiating cellular contact between leukocytes and
endothelium, thus supporting loose interactions
required for leukocyte rolling under blood-flow conditions. This has
been considered as the first step in a multistep model of leukocyte
extravasation.33 4-Integrins have been
described as being expressed by monocytes and lymphocytes. This
adhesion molecule can interact with their counterreceptors, such as
VCAM-1, under flow conditions and therefore contribute to both rolling
and firm adhesion.34 35 In contrast, other adhesion
molecules of the integrin and immunoglobulin supergene family are
unable to initiate interactions with leukocytes under wall shear
stresses >1 dyne/cm2 and seem to be specialized in the
support of adhesion strengthening and sticking in response to
activation.36 37 To assess the effect of
angiotensin II–induced upregulation of E-selectin on
leukocyte adhesion, an adhesion assay was used here that allows
quantification of leukocyte adhesion under conditions of flow. HL-60
cells were selected as a leukocyte surrogate because of their high
expression of sialyl-Lewisx–containing ligands, thus
serving as a model for E- and P-selectin–mediated
adhesion.38 In contrast to HL-60 cells, which are negative
for L-selectin and 4-integrins, these adhesion molecules
are expressed on monocytes and lymphocytes33 and can
mediate rolling under flow conditions.34 35 Since we did
not detect an upregulation of VCAM-1, which serves as a ligand for
4-integrins, this mechanism apparently does not
participate in angiotensin II–induced leukocyte adhesion.
L-Selectin ligands have been identified on high
endothelial venules and the
microvasculature.39 40 However, our results do not allow
us to conclude a contribution of L-selectin–mediated rolling due to
the effects of angiotensin II.
The results of the present study involving leukocyte adhesion support the functional significance of E-selectin expression by demonstrating an increased level of E-selectin–dependent adhesion of HL-60 cells after stimulation with angiotensin II. The specificity of this reaction was proven by adhesion blockade with anti–E-selectin antibodies. This indicates that the observed angiotensin II–induced increase in E-selectin expression is effective in increasing leukocyte adhesion under conditions of flow.
So far, two angiotensin II receptors have been cloned. The AT1 receptor is found in most tissues.41 Apart from its effects on vascular tone, it regulates the growth of vascular smooth muscle cells.42 The AT2 receptor is highly expressed in embryonic tissues, the uterus, and heart.24 It appears to mediate differentiation and apoptosis in some cell types.43 Effects of angiotensin II on endothelial PAI-I secretion and fibroblast growth that are related to angiotensin II receptors other than AT1 or AT2 have been observed.44 45 Our experimental data suggest that the expression of E-selectin by angiotensin II is modulated via AT1 receptors: only the AT1 antagonist DUP 753 blocked the angiotensin II–induced leukocyte adhesion, whereas the AT2 receptor antagonist PD 123177 did not inhibit the effects of angiotensin II significantly. The presence of AT1 receptors on coronary endothelial cells was further supported by demonstrating the mRNA of the AT1, but not the AT2, receptor by RT-PCR. However, since the specificity of the AT1- and AT2-receptor blockers toward other angiotensin II receptors is not known, the data do not exclude the participation of other not-yet-known angiotensin II receptors.
Several investigators have shown ACE inhibitors to possess protective effects regarding the development of endothelial dysfunction.9 10 29 In animal studies of cardiovascular disease, these inhibitors reduce the formation of atherosclerotic plaques in the descending thoracic aorta of the Watanabe heritable hyperlipidemic rabbit.8 In a rat model, ACE inhibitors prevented myointimal proliferation in response to injury of vascular endothelium.10 It was also shown that monocyte accumulation in the subendothelial space is reduced during ACE inhibition.46 These results are paralleled by the clinical observation of reduced cardiac ischemic events in patients treated with ACE inhibitors, which is hardly explained by their hemodynamic effects alone.7 The underlying mechanisms of these beneficial effects are only poorly understood. Apart from blocking the degradation of bradykinin, ACE inhibitors also decrease angiotensin II formation.47 Both peptides affect endothelial function and may therefore contribute to the protective effects of ACE inhibitors. ACE inhibitors markedly potentiate the release of NO in the presence of bradykinin in coronary arteries,48 which is known to inhibit leukocyte adhesion.49 A direct role of angiotensin II in the adhesion of leukocytes to coronary endothelial cells has not been demonstrated so far. The present study reveals that angiotensin II increases leukocyte adhesion to endothelial cells and thus acts in a direction opposite that of bradykinin and NO.
Increased adhesion of leukocytes to endothelial cells covering fatty streaks and an accumulation of leukocytes in the subendothelial space have been observed.2 Adhesion molecule expression is a prerequisite for the interaction of leukocytes with endothelium. Indeed, the adhesion molecules VCAM-1 and E-selectin have been shown to be present in atherosclerotic lesions,1 4 and both are involved in the adhesion of monocytes.50 Our experiments describe a possible mechanism that could explain an important role for angiotensin II in the progression of atherosclerosis. An increased expression of E-selectin induced by locally increased angiotensin II generation may lead to leukocyte rolling and increased adherence of leukocytes to the arterial wall,51 followed by emigration of leukocytes into the subendothelial space, leading to further progression of the atherosclerotic lesion. The role of the selective E-selectin stimulation by angiotensin II in the scenario of multiple stimuli for adhesion molecule expression during formation of atherosclerotic plaques requires further investigation. Furthermore, it would be interesting to determine the neovasculature responsiveness of endothelial arteriosclerotic plaques to angiotensin II and to compare this with the responsiveness of the endothelium of coronary arteries.51 52
In summary, we obtained evidence that angiotensin II enhances leukocyte adhesion by modulating the expression of E-selectin on human coronary endothelial cells. This phenomenon establishes a link between the angiotensin system, which has so far been mainly associated with regulation of vascular tone, and cellular processes, which are thought to be involved in the development of vascular remodeling and atherosclerosis.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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The excellent technical assistance of K. Vetter and S. Wentzel is acknowledged.
Received July 8, 1996; accepted August 13, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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