(Circulation. 1999;99:2317-2322.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
Androgen Exposure Increases Human Monocyte Adhesion to Vascular Endothelium and Endothelial Cell Expression of Vascular Cell Adhesion Molecule-1
From the Heart Research Institute (J.A.M., W.J., D.S.C.); the Departments of Cardiology (J.A.M., D.S.C.) and Andrology (D.J.H.), Royal Prince Alfred Hospital; and the Department of Medicine, Sydney University (D.J.H., D.S.C.), Sydney, Australia.
Correspondence to Dr Jane A. McCrohon, Department of Cardiology, Royal Prince Alfred Hospital, Missenden Rd, Camperdown 2050, Sydney, Australia. E-mail janem{at}card.rpa.cs.nsw.gov.au
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Abstract |
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Background—Male sex is an independent risk factor for coronary artery disease. Owing to the importance of monocyte adhesion to endothelial cells in the development of atherosclerosis, we hypothesized that androgens might promote this process. We therefore studied the effects of the nonaromatizable androgen dihydrotestosterone (DHT) on human monocyte adhesion to human endothelial cells and on endothelial cell–surface expression of adhesion molecules.
Methods and Results—Human umbilical vein
endothelial cells (HUVECs) were grown to confluence in
media supplemented with postmenopausal female serum, then exposed for
48 hours to either DHT (40 and 400 nmol/L), with or without the
androgen receptor blocker hydroxyflutamide (HF) (4 µmol/L); HF
alone; or vehicle control (ethanol 0.1%). Human monocytes obtained by
elutriation were incubated for 1 hour with the HUVECs at 37°C, and
adhesion was measured by light microscopy. Compared with vehicle
control, monocyte adhesion was increased in the androgen-treated HUVECs
in a dose-dependent manner (116±6% and 128±3% for DHT 40 and 400
nmol/L respectively; P<0.001). HF blocked this increase
(P
0.3 compared with control). Surface expression of
endothelial cell adhesion molecules was measured by
ELISA and revealed an increased expression of vascular cell adhesion
molecule-1 (VCAM-1) in the DHT-treated HUVECs (125±5% versus 100±4%
in controls; P=0.002), an effect also antagonized by HF
(P0.3 compared with controls). Furthermore, the
DHT-related increase in adhesion was completely blocked by coincubation
with anti–VCAM-1 antibody. Comparable results were obtained in
arterial endothelial cells and in
endothelium stimulated with the cytokine tumor
necrosis factor-
.
Conclusions—Androgen exposure is associated with increased human monocyte adhesion to endothelial cells, a proatherogenic effect mediated at least in part by an increased endothelial cell–surface expression of VCAM-1.
Key Words: hormones • atherosclerosis • cell adhesion molecules
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Introduction |
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There is an important sex difference in coronary heart disease risk, with an earlier onset of disease in males and excess mortality in men throughout adult life.1 2 Although there is a growing abundance of clinical and basic science data supporting a favorable effect of estrogen and perhaps progesterone on female cardiovascular risk and atherogenic processes,3 4 5 6 there are comparatively few data on the possible proatherogenic effects of androgens. Recent animal studies in chicks7 and cynomolgus monkeys8 support this possibility, documenting increased plaque formation associated with testosterone treatment. Few mechanistic studies, however, have examined the vascular effects of androgens in humans, although we have recently demonstrated an association between androgen deprivation and enhanced endothelial function in older men consistent with a deleterious effect of androgens on vascular reactivity.9
Monocyte adhesion to endothelial cells is an important early event in atherogenesis, controlled in part by expression of adhesion molecules on the endothelial cell surface.10 11 We therefore aimed to explore the effects of the potent nonaromatizable androgen dihydrotestosterone (DHT) on monocyte–endothelial cell adhesion and on the expression of endothelial cell adhesion molecules using primary human cells.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Materials
Phenol red–free variable amino acid RPMI cell culture medium was obtained from Life Technology. The sex-steroid hormone used was DHT (Sigma Chemical Co) because it cannot be aromatized to estrogenic metabolites. Hydroxyflutamide (HF), a nonsteroidal androgen-receptor antagonist, was a gift from Dr David Handelsman, Department of Andrology, Royal Prince Alfred Hospital, Sydney, Australia. Interleukin-1ß (IL-1ß) was obtained from Genzyme Corp. Tumor necrosis factor-
(TNF-) was obtained
from Boehringer Mannheim. Mouse anti-human monoclonal
antibodies against vascular cell adhesion molecule-1 (VCAM-1),
intercellular adhesion molecule-1 (ICAM-1), and E-selectin were
obtained from Becton-Dickinson, and isotype mouse IgG1 and IgG2 not
directed against endothelial cell antigens were
obtained from ICN Immunobiologicals. Sheep anti-mouse
antibody–horseradish peroxidase conjugate was obtained from
Amersham International.
Endothelial Cell Culture
Human umbilical vein endothelial cells (HUVECs)
were harvested enzymatically from male infant umbilical cords under
sterile conditions as described by Minter et al12 and
established as primary cell cultures in phenol red–free M199 (Gibco
Life Sciences) containing L-glutamine 2 mmol/L (ICN
Biomedicals), 0.5% endothelial cell growth promoter
(Starrate Products), penicillin 100 U/mL, streptomycin 0.1 mg/mL,
and 20% filtered human serum from healthy postmenopausal female
volunteers not taking hormone replacement therapy. Human umbilical
arterial endothelial cells (HUAECs) were
harvested and cultured by the same methods. Commercially bottled media
had been filtered at 0.1 µmol/L, and powdered medium was
reconstituted with endotoxin-free water and filtered at 0.2
µmol/L.
Endothelial cell monolayers (passages 2 to 4) were propagated on gelatin-coated flasks in phenol red–free medium, then trypsinized and replated onto gelatin-coated 24-mm-diameter tissue-culture wells or 96-well plates for monocyte adhesion and cell adhesion molecule expression studies, respectively. Wells were gelatin coated with 1 mL/5 cm2 Hemaccel (Behringwerke AG) diluted 1:250 in PBS and incubated for 1 hour at 37°C, and excess solution was removed before use. Endothelial cells were grown to confluence before sex-steroid hormone treatment and were used within 72 hours. The purity of the endothelial cell monolayers was confirmed macroscopically by their cobblestone pattern and periodically by cell staining with a monoclonal antibody specific for von Willebrand factor. At the end of the 48-hour treatments, cell viability was >95% (by Trypan blue exclusion) for each condition.
Isolation of Human Monocytes
White cell concentrates (Red Cross Blood Bank) were obtained
from the peripheral blood of healthy human volunteers, and
monocytes were removed within 24 hours of collection by density
gradient separation of the white cells on Lymphoprep (Nycomed Pharma)
followed by counterflow centrifugation elutriation at
20°C, as previously described by our group,13 14 by use
of a Beckman J2–21 M/E centrifuge equipped with a JE-6B
elutriation rotor and a 4.2-mL elutriation chamber (Beckman
Instruments, Inc). The elutriation buffer was HBSS without calcium or
magnesium (Sigma) supplemented with EDTA (0.1 g/L) and 1%
heat-inactivated human serum. The system and tubing were
rinsed with 250 mL each of 70% ethanol, endotoxin-free water, 6%
hydrogen peroxide, endotoxin-free water, and elutriation buffer in that
order before the Lymphoprep-derived mononuclear cell fraction was
loaded at 9 mL/min into the elutriation rotor chamber (2020 rpm at
20°C). Flow rate was increased by 1-mL/min increments every 10
minutes, and monocytes were typically eluted at between 16 and 17
mL/min. Collected fractions were examined by a Cytospin system
(Shandon) and Wright's stain (Diff-Quik, Laboratory-Aids). Monocyte
purity of >90% and viability of >95% by Trypan blue exclusion were
confirmed on light microscopy, and the monocytes were resuspended in
RPMI containing 2% human serum and used immediately for adhesion
studies.
Monocyte–Endothelial Cell Adhesion Assay
Endothelial cells were propagated for 1
passage in phenol red–free RPMI supplemented with 20% human
postmenopausal female serum from healthy donors. This serum was used
because of the low baseline levels of sex-steroid hormones (estradiol
<75 pmol/L, progesterone <0.8 nmol/L, and testosterone <2 nmol/L).
Confluent endothelial monolayers were established in
24-mm-diameter wells before incubation for 48 hours with the following
treatments: (1) control wells treated with 0.1% ethanol (used to
dissolve the sex-steroid hormones); (2) DHT 40 nmol/L; (3) DHT 400
nmol/L (the normal range of testosterone in human male serum is 4 to 40
nmol/L); (4) DHT 40 nmol/L and HF 400 nmol/L; (5) DHT 400 nmol/L and HF
4 µmol/L; and (6) HF 4 µmol/L. Each treatment group was
divided after 24 hours of hormone treatment into basal and stimulated
states, the latter receiving IL-1ß (50 U/mL) or TNF- (500 U/mL)
for the final 24-hour period. Separate adhesion experiments were
performed 5 times for control and DHT-treatment groups and 3 times for
HF exposure. Each experiment used at least triplicate wells for each
condition. An additional series of experiments investigated the
interaction of DHT and IL-1ß in terms of stimulating cell adhesion
and involved sequential and/or coincubation conditions, as described in
the Results section.
The adhesion assay involved the addition of 1.5x106 monocytes per milliliter of RPMI/2% human serum to the endothelial monolayer and incubation for 1 hour at 37°C under 5% CO2 in air. After 1 hour, nonadherent cells were removed by gentle washing with a 1000-µL automatic pipette (Gilson), and the 1-mL suspension was stored on ice until the cell concentration was determined with a Neubauer hemocytometer (Weber Scientific). The initial suspensions and the suspension from each well were counted 4 times by an observer blinded to the treatment conditions. The percentage of adherent monocytes was calculated by comparison with the initial concentration. This method has been shown to have a low intraobserver error, with a coefficient of variation of <5% and maximal basal adhesion after 1 hour of incubation.13 Basal monocyte–endothelial cell adhesion in these experiments was 25±5%, increasing with IL-1ß stimulation to 55±6% (P<0.01).
Endothelial Cell Adhesion Molecule
Expression
The cell-surface expression of adhesion molecules on the
endothelial cell monolayers exposed to different
treatments was assessed with an ELISA technique. Confluent cell
monolayers were established in 96-well plates and, as was done for the
adhesion studies, exposed for 48 hours to the control or hormone
treatments as outlined above, with or without IL-1ß stimulation (50
U/mL) for the last 24 hours of treatment. Wells were then washed twice
with HBSS; monoclonal antibodies to ICAM-1, VCAM-1, E-selectin, and
isotype mouse IgG (0.1 µg in 100 µL of HBSS with 10%
heat-inactivated human serum) were added and left for 30
minutes. The layers were washed 3 times with HBSS and 0.05% Tween 20
before a 30-minute incubation with sheep anti-mouse
antibody/horseradish peroxidase conjugate (1:500 in 100 µL of HBSS
with 10% heat-inactivated human serum and 0.05% Tween
20). After an additional 4 washes, 150 µL of ABTS substrate
(Kirkegaard and Perry Laboratories) was added to each well and allowed
to develop for 15 minutes. Results were expressed as units of optical
density measured at 414 nm with an ELISA plate reader (Titretek
Multiscan, Flow Laboratories).
Adhesion Assay With Neutralizing Antibodies to Cell Adhesion
Molecules
Endothelial cells grown to confluence and
exposed to different hormone conditions (as described above) were
incubated with human monoclonal antibodies (2 µg/mL) to either
ICAM-1, VCAM-1, E-selectin, or all 3 for 60 minutes at 37°C, as
described by Meng et al.15 The cells were then washed 3
times with RPMI immediately before the addition of the monocyte
suspension. Adhesion was then measured as before, and each treatment
group was compared with a baseline state in which no antibodies had
been added to assess the relative importance of each cell adhesion
molecule under the different treatment conditions.
Statistical Analysis
All descriptive data are expressed as mean±SEM, and the data
were analyzed on SPSS for Windows 6.0. Because each experiment
involved both endothelial cells and monocytes from
different donors, results for the adhesion assays and ELISAs for cell
adhesion molecule expression were expressed as a percentage of the
control condition within each experiment. Groups were compared by 1-way
ANOVA followed by the Student-Newman-Keuls test for multiple
comparisons and independent-samples t tests for comparisons
between groups. Statistical significance was inferred at a 2-sided
value of P<0.05.
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Results |
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Monocyte-Endothelial Cell Adhesion and Expression of Endothelial Cell Adhesion Molecules
Androgen exposure was associated with an increase in monocyte adhesion to IL-1ß–stimulated endothelial cell monolayers (Figure 1
). This was dose
related (116±6% and 128±3% for DHT 40 nmol/L and 400 nmol/L,
respectively, compared with control; P<0.001 by ANOVA and
P<0.01 for each DHT concentration compared with control)
(Figure 2) and was not observed in basal
state (non–IL-1ß–stimulated) endothelium (data not
shown). This androgen-mediated increase in adhesion was abolished by
cotreatment with the androgen-receptor antagonist HF
(95±8% and 105±4% for DHT 40 nmol/L-HF 400 nmol/L and DHT 400
nmol/L-HF 4 µmol/L, respectively; P>0.3 compared
with control). HF alone did not affect adhesion (94±6% compared with
controls; P=0.4). These results suggest an androgen-mediated
amplification of monocyte–endothelial cell adhesion
that is at least partially receptor dependent.
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The effect of androgen treatment on endothelial
cell–surface adhesion molecule expression was then studied with an
ELISA technique. Treatment of endothelial cell
monolayers for 24 hours with IL-1ß increased cell-surface expression
of all 3 cell adhesion molecules: ICAM-1 (optical density, 0.55±0.02
versus 0.98±0.02; P<0.001), VCAM-1 (optical density,
0.22±0.01 versus 0.48±0.03; P<0.001), and E-selectin
(optical density, 0.12±0.01 versus 0.30±0.02; P<0.02). In
the stimulated state, there was an increase in
endothelial surface expression of VCAM-1 with DHT
compared with control wells (DHT 40 nmol/L 125±5% and DHT 400 nmol/L
123±7% versus control 100±4%; P<0.01 for each
comparison) (Figure 3). Furthermore, this
increase in VCAM-1 expression was reduced to control levels in HUVECs
treated with both DHT and HF (97±6% compared with control;
P=0.7). HF alone did not alter VCAM-1 expression (105±7%
versus control; P=0.5). There was no association between
treatment condition and surface expression of either ICAM-1 or
E-selectin (data not shown). In the basal state (without IL-1ß),
surface expression of VCAM-1, ICAM-1, and E-selectin did not change
significantly between treatment conditions, similar to the results of
the adhesion studies described above. In all experiments, there was no
significant binding of isotype IgG to endothelial cells
in either the basal or stimulated state.
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These results were derived from endothelial cells coincubated with IL-1ß during the final 24 hours of treatment exposure. To further explore the interaction between DHT and IL-1ß, similar experiments were performed with sequential exposures in which (1) DHT was washed off the endothelial cells completely after 48 hours, followed by a subsequent 24-hour exposure to IL-1ß, or (2) IL-1ß was added to the media for 24 hours (and then washed off) before a 48-hour exposure to DHT (that is, the reverse of experiment 1 above). In both cases, the IL-1ß– and DHT-exposed HUVECs demonstrated increased adhesion and increased VCAM-1 expression compared with DHT-vehicle (ethanol 0.1%)–treated controls similarly exposed to IL-1ß. In experiment 1, DHT exposure before IL-1ß increased adhesion (DHT 400 nmol/L 131±4% versus 100±2% for controls; P=0.03) and VCAM-1 expression (DHT 400 nmol/L 110±3% versus 100±2% for controls; P=0.03). In experiment 2, IL-1ß exposure before DHT treatment also increased adhesion (DHT 400 nmol/L 124±3% versus 100±1% for controls; P=0.03) and VCAM-1 expression (DHT 400 nmol/L 131±6% versus 100±7% for controls; P=0.01). In each case, HF antagonized the effect of DHT (data not shown).
To demonstrate whether these effects would also be observed with arterial endothelial cells, additional experiments were performed with HUAECs. DHT-treated HUAECs were similarly associated with an increase in monocyte adhesion after IL-1ß stimulation (DHT 400 nmol/L 131±7% versus 100±7% for controls; P<0.03), and this effect could be blocked by coincubation with HF (DHT 400 nmol/L-HF 4 µmol/L 88±4%; P=0.1 compared with control). Furthermore, as was seen in the equivalent HUVEC experiments, this androgen-mediated increase in monocyte–endothelial cell adhesion was associated with an increase in VCAM-1 expression (DHT 400 nmol/L 116±5% versus 100±1% for controls; P=0.04), which was reduced by coadministration of HF (107±3%; P=0.1 compared with control).
To demonstrate that this effect was not confined to
endothelium costimulated with DHT and IL-1ß,
experiments were performed with another cell-adhesion–promoting
cytokine, TNF-. DHT-treated HUVECs stimulated with TNF-
also showed increased monocyte adhesion (DHT 400 nmol/L 130±1% versus
100±2% for controls; P=0.002) and VCAM-1 expression (DHT
400 nmol/L 114±2% versus 100±4% for controls; P=0.02).
As seen with IL-1ß stimulation, the androgen-mediated increase in
adhesion and VCAM-1 expression observed in TNF-–stimulated
endothelium could be antagonized by HF (100±5% and
103±8% for adhesion and VCAM-1 expression, respectively;
P>0.5 compared with control). E-selectin and ICAM-1
expression were not significantly different between conditions
(102±6% and 101±4% for E-selectin and ICAM-1, respectively, after
androgen exposure; P>0.5 compared with control).
Neutralizing Antibodies to Cell Adhesion Molecules
The ELISA results showing increased surface expression of VCAM-1
on HUVECs treated with androgen were confirmed by repeat adhesion
studies involving a 60-minute incubation of the
endothelial monolayers with antibodies to VCAM-1,
ICAM-1, and E-selectin before the addition of human monocytes. In these
experiments, VCAM-1 antibody effectively eliminated the
androgen-related increase in monocyte–endothelial cell
adhesion (DHT 400 nmol/L 91±6% compared with control 97±5%;
P=0.4) (Figure 4). ICAM-1
antibody reduced overall monocyte adhesion compared with baseline but
did not change the relative increase seen between control and DHT
treatment (DHT 400 nmol/L 113±8% compared with control 75±4%;
P=0.002); results were similar for E-selectin (DHT 400
nmol/L 118±6% compared with control 93±8%; P=0.02). As
expected, incubation with all 3 cell adhesion molecule antibodies
greatly reduced overall adhesion and eliminated any difference between
control and androgen-treated wells (DHT 400 nmol/L 74±4% compared
with control 75±3%; P=0.9).
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0.02 and **P=NS comparing control
with DHT. |
Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The effect of androgens on different aspects of atherogenesis has received little attention despite the marked male predisposition to occlusive vascular disease.1 2 In the present study, we have demonstrated that androgen exposure leads to a dose-related and receptor-mediated increase in human monocyte adhesion to endothelial cells, a key early event in atherosclerosis. This effect is mediated at least in part by an androgen receptor–dependent increase in endothelial cell expression of the important adhesion molecule VCAM-1.
A proatherogenic effect of androgens is supported by recent work in experimental animals. For example, Adams et al8 documented an approximate doubling of coronary artery plaque size in female postmenopausal cynomolgus monkeys treated with testosterone and a cholesterol-enriched diet, and Hutchison et al16 documented arterial endothelial dysfunction in hypercholesterolemic rabbits that were administered androgens. Similar data are not available in humans.
The incidence of coronary deaths in men aged 35 to 64 years
exceeds that in age-matched females by up to 500%.17
Within each gender, however, there has been no consistent
association between androgen levels and cardiovascular
event rates. Although most cross-sectional data suggest either no
effect or an inverse correlation between serum testosterone levels and
cardiovascular event rates in men,18 19 20 21
prospective studies have not shown a significant
correlation.22 23 24 25 26 27 Therefore, although small
physiological variations in androgen levels within
genders may not correlate clearly with patterns of
atherosclerosis, it is likely that the large difference
in androgen levels and receptors between genders is a significant
contributor to the sex difference in cardiovascular
risk. In the body, testosterone is partially metabolized by aromatases
to estrogen and by 5-reductase to DHT. Testosterone may therefore
not be a clear marker of androgen action owing to its estrogenic
metabolites. In the present study, the androgen DHT was used to
assess the effect of androgens on monocyte–endothelial
cell adhesion for several reasons: it is nonaromatizable, thus avoiding
confounding estrogenic effects, and it is one of the most potent
androgens in androgen-sensitive tissues, binding to the cytoplasmic
androgen receptor 2 to 10 times more avidly than
testosterone.28
In the present study, DHT-treated endothelial cells showed an increased surface expression of VCAM-1, which suggests increased production and/or recruitment of VCAM-1 to the endothelial cell surface. In concordance with the functional adhesion data, this androgen-mediated increase in surface expression of VCAM-1 was abrogated by HF, which indicates that these processes are mediated at least in part via androgen receptors. Interestingly, 17ß-estradiol, the potent estrogen, decreases endothelial cell adhesion molecule expression and monocyte adhesion, also via its sex-steroid receptor.29 The intracellular events accounting for such changes require further study; however, receptor-activated stimulation of gene transcription is a likely mechanism. Of note, the endothelial cells treated with androgen in the current experiments were coincubated with whole human serum. This suggests that androgen-mediated monocyte–endothelial cell adhesion is a physiologically relevant event, even in the presence of lipoproteins such as HDL, which have been shown to be protective against cell adhesion molecule expression.30
Our experiments involved an in vitro model of HUVECs and monocytes
obtained from peripheral blood by elutriation, which may
differ from the in vivo situation. Endothelial cells in
vivo do not usually express high levels of ICAM-1, VCAM-1, or
E-selectin,31 32 whereas the HUVECs used in our
experiments did express these adhesion molecules, presumably owing to
the tissue-culture environment. This may be similar to the situation
seen in atherosclerosis in which cytokines such
as IL-1ß and TNF- are present and endothelial
expression of adhesion molecules is stimulated.31 33 34 In
this context, the androgen-related increase in cell adhesion observed
after stimulation with IL-1ß or TNF-, as well as the similarity of
findings with both venous and arterial
endothelial cells in the present experiments, is
consistent with potentially important effects in vivo at
arterial sites prone to
atherosclerosis.
The interaction between androgen and cytokine required to promote monocyte–endothelial cell adhesion did not require coincubation with DHT and IL-1ß and was observed regardless of the order of DHT or IL-1ß stimulation. Although the exact intracellular mechanism of this interaction is not known, these data suggest that the sequential or simultaneous presence of androgen and cytokine might promote monocyte adhesion in the in vivo situation.
In the present study, we observed an androgen-related increase in
endothelial cell expression of the adhesion molecule
VCAM-1. Recent reports have confirmed an important role for VCAM-1 in
atherogenesis.31 35 Studies have demonstrated a
significant upregulation of VCAM-1 in plaques found in diet-induced
atherosclerosis in animal models36 and in
human atherosclerotic lesions.31 37 In contrast to other
adhesion molecules, VCAM-1 in isolation is able to mediate leukocyte
adhesion via integrin interaction,38 whereas E-selectin
and ICAM-1 can mediate only part of the complex adhesion
process.39 In addition, VCAM-1 expression precedes and is
correlated with the degree of macrophage accumulation in human
plaques. In our experiments, IL-1ß–induced adhesion of monocytes to
endothelium was only reduced by 
30% in the presence
of anti–VCAM-1, anti–ICAM-1, and anti–E-selectin antibodies, which
suggests either incomplete blockade of these adhesion molecules or the
coexistence of other important proadhesion factors not blocked in our
study. These might include surface-associated chemokines such as
monocyte chemoattractant protein, growth-regulated protein, and
monocyte colony stimulating factor, as well as other,
as-yet-unidentified adhesion molecules.40
In summary, DHT increases human monocyte adhesion to vascular endothelium, at least in part through an androgen receptor–mediated effect on endothelial expression of VCAM-1. This androgen-mediated increase in monocyte adhesion may be an important mechanism in the greater susceptibility of men to the development of premature atherosclerosis.
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Acknowledgments |
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Dr McCrohon is supported by Sydney University and the Department of Cardiology, Royal Prince Alfred Hospital; Dr Jessup by The National Health and Medical Research Council; and Dr Celermajer by The Medical Foundation, University of Sydney. We are thankful to S. Nakhla, Dr M.R. Adams, and Dr K.K. Stanley for their technical advice and assistance.
Received August 27, 1998; revision received December 8, 1998; accepted December 29, 1998.
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