© 1999 American Heart Association, Inc.
Integrative Physiology |
Estradiol Inhibits Leukocyte Adhesion and Transendothelial Migration in Rabbits In Vivo
Possible Mechanisms for Gender Differences in Atherosclerosis
From the Departments of Obstetrics and Gynecology (L.N., S.P., R.S., G.C.) and Molecular and Medical Pharmacology (R.S., G.C.), UCLA School of Medicine, University of California, Los Angeles, Calif, and Department of Pathobiology and Program in Nutritional Sciences (M.R.), University of Washington, Seattle, Wash.
Correspondence to Gautam Chaudhuri, MD, PhD, Departments of Obstetrics and Gynecology, and Molecular and Medical Pharmacology, UCLA School of Medicine, 10833 LeConte Ave, 27-139 CHS, University of California, Los Angeles, CA 90095-1740. E-mail gchaudhu{at}obgyn.medsch.ucla.edu
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Abstract—The mechanism by which estrogens protect against atherosclerosis is not known. We evaluated in vivo whether there is a gender difference in monocyte adhesion and subendothelial migration in hypercholesterolemic rabbits and whether any gender differences observed are due to estradiol. Monocyte adhesion and subendothelial migration were assessed in a blinded fashion by analyzing a standardized segment of aorta using a scanning electron microscope. We also assessed whether estradiol modulates induction of vascular cell adhesion molecule-1 (VCAM-1) protein using Western blot and flow cytometric analyses. We observed that male rabbits develop more monocyte adhesion and subendothelial migration than do female rabbits during hypercholesterolemia. We also observed that oophorectomized rabbits given physiological estradiol supplementation demonstrate fewer adherent and subendothelial monocytes than do oophorectomized rabbits given placebo. VCAM-1 protein expression was increased in aortae obtained from hypercholesterolemic, oophorectomized animals supplemented with placebo, and this increase was attenuated by estradiol. Finally, in cultured rabbit aortic endothelial cells stimulated with lysophosphatidylcholine, we observed an increase in VCAM-1 protein that was inhibited in a concentration-dependent fashion by estradiol. We have demonstrated in vivo that there is a gender difference in monocyte adhesion to endothelial cells and transendothelial migration after hypercholesterolemia and that this gender difference is due in part to estradiol. Our results also suggest that estradiol inhibits monocyte adhesion by inhibiting expression of VCAM-1.
Key Words: estrogen • estradiol • atherosclerosis • vascular cell adhesion molecule-1 • monocyte adhesion
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Introduction |
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Women in the reproductive age group are at lower risk for developing cardiovascular disease (CVD) as compared with men of similar age.1 2 This gender difference has been well documented in both human and animal studies.2 3 4 5 6 7 Hamm et al5 demonstrated that in nonhuman primates there was significantly more coronary artery stenosis in males than in females in response to an atherogenic diet. Furthermore, they found that differences in lipid levels did not account for this influence of gender in the development of CVD. Similar observations have been made in male and female rabbits after administration of a cholesterol (2%)–enriched diet.6 The protection against CVD observed in females has been ascribed to estrogen. Estrogen replacement therapy in postmenopausal women reduces the risk of CVD relative to the risk in postmenopausal women who do not receive estrogen replacement.7 8 In support of these clinical observations, Adams et al9 observed that ovariectomy in nonhuman primates resulted in a doubling of the extent of atherosclerosis in animals fed an atherogenic diet compared with a similar group of animals receiving estrogen replacement. Adams et al10 also demonstrated that in animals fed an atherogenic diet, administration of 17ß-estradiol (E2) resulted in the formation of a plaque area that was 50% smaller than that seen in animals given no hormone replacement. Studies in oophorectomized rabbits also showed that estrogen administration attenuated aortic accumulation of cholesterol11 and reduced the degree of atherosclerosis after an atherogenic diet.12 However, the mechanisms by which estrogens protect against atherosclerosis are not known.
The adhesion of monocytes to endothelial cells coupled with transendothelial migration are essential components of an inflammatory response and occur continuously throughout the entire atherogenic process.13 We therefore decided to assess whether there was a gender difference in the number of monocytes that both adhere to and migrate across the endothelial lining of the aorta in vivo in rabbits fed a cholesterol-enriched (0.5%) diet and whether E2 was responsible for this difference. To further elucidate possible mechanisms by which E2 may prevent monocyte adhesion, we also assessed the role of E2 in inhibiting expression of vascular cell adhesion molecule-1 (VCAM-1). The rabbit was selected as the animal model, because the female of this species is in persistent estrus14 ; therefore, the animals are under the constant influence of E2 with no compounding influence of progesterone. Furthermore, we took advantage of previous studies of the time course of lesion development in hypercholesterolemic rabbits15 to focus this investigation on those time points at which monocyte adhesion and subendothelial migration are known to first occur.
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Materials and Methods |
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Materials
Rabbit chow (modified laboratory rabbit diet 5321, with 0.5% cholesterol) was purchased from Purina Mills, Inc. E2 pellets were purchased from Innovative Research of America. Hybond-polyvinylidene difluoride (PVDF) membrane, enhanced chemiluminescence reaction detection reagent, and autoradiography film (Hyperfilm) were obtained from Amersham Life Science, Inc. Collagenase was obtained from Gibco-BRL. Plastic plates for cell culture were obtained from Costar. Charcoal-extracted FBS was obtained from Hyclone Laboratories, Inc. The GeneAmp RNA PCR kit was obtained from Perkin-Elmer. TA vector pCR 2.1 was obtained from Invitrogen. Goat F(ab')2 anti-mouse IgG FITC conjugate was obtained from Caltag Laboratories. Gelatin for coating the culture plates and all other reagents were obtained from Sigma Chemical Co. ICI-182780 was supplied by Dr. G. Rubanyi (Berlex Biosciences, Richmond, Calif). VCAM-1 antibody for cell culture studies was supplied by Dr M.I. Cybulsky (Brigham and Women's Hospital, Boston, Mass) and for Western blot was obtained from Santa Cruz Biotechnology, Inc. GAPDH monoclonal antibodies were obtained from Chemicon.
Animals
Male and female New Zealand White (NZW) rabbits weighing 3 to
3.5 kg were used. All animals were initially fed regular chow for 2
weeks. Certain animals then underwent oophorectomy (OVX) with placement
of either placebo or E2 pellets (1.5 mg, 60-day
release). Briefly, animals were placed under anesthesia
with ketamine and halothane. With a sterile technique, a
midline abdominal incision was made, and the ovaries were identified
and then excised after ligation of the ovarian artery and vein.
The abdominal incision was then closed in layers with delayed
absorbable suture. A 1-cm skin incision was then made at the nape of
the neck, and the placebo or E2 pellets were
placed subcutaneously. The skin was closed with delayed absorbable
sutures. All animals received prophylactic antibiotics
(cefazolin, 500 mg IM). The animals were allowed to recover from
surgery for the next 7 to 10 days, during which time they were fed
normal chow (NC). They were then continued on NC or placed on a 0.5%
cholesterol diet (HC) for 4 weeks. Animals developing
evidence of postoperative infection were excluded from the study. An
aliquot of blood was collected into tubes containing EDTA from each
animal on a weekly basis from a peripheral ear vein to
assess lipid and E2 levels. At the end of the
4-week feeding period, animals were placed under deep
anesthesia with ketamine (10 mg/kg, IM) and
xylazine (40 mg/kg, IM) in preparation for perfusion fixation of the
aortic tree. Certain animals were euthanized by exsanguination without
perfusion fixation so that aortic tissue could be harvested for Western
blotting. The animal protocol was formally approved by the Animal
Research Committee of the University of California.
Experimental Protocols
Protocol 1: Gender Differences in Monocyte Adhesion in Gonad-Intact
Rabbits Fed Normal or Cholesterol-Enriched Chow
Animals were divided into the following 4 groups: (1)
gonad-intact females fed NC for 4 weeks (n=5), (2) gonad-intact males
fed NC for 4 weeks (n=5), (3) gonad-intact females fed HC for 4 weeks
(n=8), and (4) gonad-intact males fed HC for 4 weeks (n=8).
Protocol 2: Effects of E2 Supplementation on Monocyte
Adhesion in Animals Fed a Cholesterol-Enriched Diet
Animals were divided into the following 2 groups: (1) OVX
females supplemented with placebo and fed HC for 4 weeks (n=7) and (2)
OVX females supplemented with E2 and fed HC for 4
weeks (n=6).
Protocol 3: Effects of E2 Supplementation on VCAM-1
Protein Expression in Animals Fed a Cholesterol-Enriched Diet
Animals were divided into the following 3 groups: (1) OVX
females supplemented with placebo and fed NC for 4 weeks (n=5), (2) OVX
females supplemented with placebo and fed HC for 4 weeks (n=5), and (3)
OVX females supplemented with E2 and fed HC for 4
weeks (n=5).
Protocol 4: Effects of E2 on VCAM-1 Protein Expression
in Cultured Endothelial Cells Stimulated With
Lysophosphatidylcholine (Lyso-PC)
The cell groups were as follows: (1) cells incubated with
vehicle, (2) cells incubated with vehicle for 48 hours followed by
stimulation with 100 µmol/L lyso-PC for 15 hours (10 mmol/L
stock in ethanol), (3) cells incubated with 1000 pg/mL
E2 for 48 hours followed by stimulation with
100 µmol/L lyso-PC for 15 hours, and (4) cells incubated with
3000 pg/mL E2 for 48 hours followed by
stimulation with 100 µmol/L lyso-PC for 15 hours. Before
treating the cells, the presence of estrogen receptors on the cells was
confirmed with the use of reverse transcription–polymerase chain
reaction (RT-PCR) as described below. In some experiments, ICI-182780
(9 ng/mL), an estrogen receptor antagonist,16
was added in the culture medium just before addition of 3000 pg/mL
E2. Cells were then prepared for
fluorescence-activated cell sorting (FACS) as described
below.
Perfusion Fixation
Perfusion fixation was carried out according to the method
described by Rosenfeld et al.15 After establishment of
deep anesthesia, one of the femoral veins was exposed and
cannulated for perfusion runoff. A cannula connected to a perfusion
apparatus was inserted into the right carotid artery. One
of the femoral arteries was also cannulated for placement of a pressure
manometer to measure mean arterial perfusion pressure.
Immediately before exsanguination, the animals were given a bolus
injection of sodium pentobarbital, and the blood was flushed out with
lactated Ringer's solution at a flow rate of 
100 mL ·
min–1 · kg–1 body
weight to yield an average intra-arterial perfusion
pressure of 100 mm Hg. When the runoff was clear of blood, 2 to 4
liters of 2.5% glutaraldehyde in 0.1N phosphate buffer
was perfused through at the above flow rate. After perfusion, the
femoral vein and carotid artery were clamped, and the arteries were
fixed under pressure for an additional 30 minutes. After perfusion
fixation, the entire aorta and common iliac arteries were dissected
from each animal. The vessels were cleaned of adherent fat and fascia,
and a 1-cm area of aorta surrounding the celiac bifurcation was
isolated and prepared for scanning electron microscopy.
Calculation of Lipid Exposure
To assure that differences in cholesterol exposure
during the feeding period could not account for differences in monocyte
adhesion between the groups, cholesterol exposure during
the 4-week feeding period was calculated for each animal and compared
with that of the other animals in the respective groups. This was done
by calculating the total cholesterol, HDL, and LDL areas
under the curve (AUCs) for each animal, with the following formula:
AUC=(Y0/2)+Y1+Y2+Y3+(Y4/2),
where Y0, Y1, Y2, Y3, and
Y4 were total cholesterol, HDL, and LDL levels
at weeks 0, 1, 2, 3, and 4, respectively. Only tissues from those
animals with comparable AUCs were selected for assessment of monocyte
adhesion and expression of VCAM-1 protein.
Tissue Preparation for Scanning Electron Microscopy
The segments of aorta surrounding the celiac bifurcation
were opened lengthwise and pinned out on
polytetrafluoroethylene. The tissue was
washed in buffer, postfixed in 1% OsO4 in PBS
for 3 hours, followed by 1 hour in 1% thiocarbohydrazide and finally
again in 1% aqueous OsO4 for 2 hours, all at
room temperature. The tissue was next dehydrated in ethanol, critical
point-dried with CO2 and sputter-coated with a
300-Å layer of gold palladium. A standardized area of each specimen
measuring 13.5 mm2 (Figure 1A
) was then examined in a blinded
fashion with a JEOL 35C scanning electron microscope at 15 kV (JEOL,
Inc). The numbers of both adherent monocytes (Figure 1B, 1D, and 1F) and monocytes covered by endothelium (Figure 1C, 1E, and 1G) were counted.
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Western Blot
After exsanguination, the entire aorta and iliac arteries were
dissected out from the animal and cleared of adherent fat and fascia.
The abdominal aorta, including the renal artery branch points, was then
isolated, and VCAM-1 and, in some experiments, GAPDH were then
analyzed by Western blot with a polyclonal goat, anti-human
VCAM-1 and mouse monoclonal anti-rabbit GAPDH antibody. The tissues
were homogenized in 50 mmol/L
tetraethylammonium-HCl (pH 7.4) containing
(in mmol/L) EGTA 0.1, EDTA 0.1, and DTT 0.5, as well as 1
µmol/L pepstatin A and 2 µmol/L leupeptin, at 4°C with the
aid of a tissue grinder fitted with a
polytetrafluoroethylene pestle. Protein
concentration was measured by the Bradford Coomassie brilliant blue
method as described by Bio-Rad Laboratories. The samples were then
diluted in loading buffer (10% glycerol, 2% SDS, and 62.5 mmol/L
Tris-HCl [pH 6.8], 5% ß-mercaptoethanol, and 0.1% bromphenol
blue). Fifty micrograms of protein in tissue homogenates
was loaded in each lane, separated on 7.5% SDS-polyacrylamide
gels, and transferred to Hybond PVDF membrane
electrophoretically using a semidry transfer system (Pharmacia
Biotech). Equal loading in each lane was confirmed by staining the PVDF
membrane with Ponceau S solution (Sigma). The membrane was blocked for
2 hours at room temperature in PBS containing 0.1% Tween-20 and 10%
nonfat milk. The membrane was incubated with VCAM-1 antibody at 1:1000
dilution for 2 hours and was washed 3 times for 15 minutes each with
wash buffer (PBS containing 0.1% Tween-20). After washing, the
membrane was incubated for 1 hour at room temperature with a
horseradish peroxidase–conjugated secondary antibody at 1:2000
dilution. The membranes were again washed 3 times in wash buffer, after
which they were incubated for 2 minutes at room temperature in
chemiluminescence reaction detection reagents. The membranes were then
exposed to autoradiography film (Hyperfilm), and the
relative intensities of the bands were quantified by
densitometric analysis (Personal Densitometer SI, Molecular
Dynamics). For reprobing for GAPDH, the membranes were stripped at
50°C for 30 minutes in buffer containing 2% SDS, 62.5 mmol/L
Tris-HCl (pH 6.8), and 100 mmol/L ß-mercaptoethanol. The
membranes were immunoblotted with GAPDH antibody at 1:5000
dilution.
Cell Culture
Rabbit aortic endothelial cells (RAECs) were
isolated from NZW rabbits fed NC. The entire descending thoracic aorta
was excised, cleaned, and incubated with 0.08% collagenase
at 37°C for 15 minutes. The aorta was rinsed with PBS, and the
endothelial cells were flushed out and seeded in
plastic plates precoated with 0.1% gelatin and cultured in DMEM-low
glucose supplemented with 10% FBS, 12.5 µg/mL
endothelial cell growth supplement, 25 U/mL heparin,
100 U/mL penicillin, and 100 µg/mL streptomycin. Culture plates with
90% confluent endothelial cells were subsequently
trypsinized with 0.25% trypsin-EDTA for 5 minutes and reseeded in
gelatin-coated (0.1%) plates, and cells from the third passage were
used for experiments. Cells were identified as
endothelial by their characteristic cobblestone
morphology. Before the cells were used, they were incubated with
E2 for 48 hours and tested for the presence of
estrogen receptors with RT-PCR as described below. Cells were placed in
medium containing phenol red–free DMEM and charcoal-extracted FBS.
Cells were then treated as described in protocol 4.
Reverse Transcription–Polymerase Chain Reaction (RT-PCR)
RAECs (106 cells) were harvested, and
total RNA was extracted using Tri-Reagent as described by the
Molecular Research Center. Reverse transcription was performed with 3
µg of total RNA, 50 units of Moloney murine leukemia reverse
transcriptase, and 100 pmol of oligo-dT and running the reaction at
42°C for 20 minutes. The resulting cDNA samples were PCR-amplified
using the GeneAmp RNA PCR kit and 100 ng each of
estrogen receptor sense and antisense primers in a 100-µL reaction
mixture. The amplification procedure consisted of initial heat
denaturation at 94°C for 5 minutes followed by PCR amplification of
estrogen receptor cDNA for 40 cycles each of denaturation at 94°C for
30 seconds, primer annealing at 55°C for 30 seconds, and extension at
72°C for 1 minute. The following set of primers was used for estrogen
receptor: sense, 5'-AGGAAGAGCTGCCAGGCCT-3', and antisense,
5'-CCAGTTGATCATGTGAACCAG-3'.17 The primers were obtained
from Custom Primers (Gibco-BRL). The amplified DNA fragment obtained
was 381 bp, as expected. The PCR product was cloned into TA vector
pCR 2.1, and its identity as estrogen receptor was confirmed by DNA
sequencing (data not shown).
Fluorescence-Activated Cell Sorting (FACS)
After various incubations as described in protocol 4, the cells
were trypsinized, collected in tubes, and washed 3 times with PBS.
Cells (5x105) were resuspended in 50 µL PBS.
Rabbit VCAM-1 mouse monoclonal antibody (Rb 1/9; 2 µg) was added to
each tube, which was kept shaking for 2 hours on ice. The cells were
washed with cold PBS 4 times and incubated with FITC-conjugated
goat F(ab')2 anti-mouse IgG at a dilution of 1:25
in PBS for 1 hour in ice and covered from light. After washing 4 times
with PBS, the cells were fixed with 1%
paraformaldehyde and analyzed by FACScan
(Becton Dickinson) analysis. Results were plotted as intensity
of fluorescence versus cell numbers.
Lipid Assays
Plasma total cholesterol, HDL, and LDL were measured
in each animal. All samples were analyzed in triplicate using
enzymatic colorimetric assays as described
previously.18 19 20 The assays were performed in the
Molecular Biology Institute Lipid and Lipoprotein Laboratory
(University of California, Los Angeles, CA). This laboratory is
certified by the Centers for Disease Control and Prevention–National
Heart, Lung and Blood Institute Lipid Standardization Program and meets
their criteria for precision and accuracy.
Radioimmunoassay for E2
The plasma concentrations of E2 were
measured by radioimmunoassay as previously described.21
Briefly, 0.6 to 0.8 mL plasma containing known amounts (400 cpm) of
[3H]E2 were extracted
with 7 mL fresh dimethyl ether, and the extract was evaporated to
dryness. The dried plasma extract was dissolved in isooctane and then
applied to a column of infusorial earth (Celite) for
chromatographic separation of E2, as
described by Brenner et al.22 Steroid fractions collected
from the column were dried and reconstituted with assay buffer for
radioimmunoassay. The results showed that peaks of radioactive and
immunoreactive estrogens coincided in Celite column
fractions.21
Statistics
Values are expressed as mean±SEM. Comparisons of means were
made by using the Student t test for unpaired values; when
more than 2 means were compared, an ANOVA with repeated measurements
was used. If a significant F value was found, the Scheffé test
for multiple comparisons was used to identify differences among groups.
Values were considered significant when P<0.05.
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Results |
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Protocol 1: Gender Differences in Monocyte Adhesion in Gonad-Intact Rabbits Fed Normal or Cholesterol-Enriched Chow
The mean cholesterol AUCs for gonad-intact female and male rabbits fed NC for 4 weeks were 482±131 and 277±94 mg/dL, respectively, and were not significantly different from one another (P>0.5). Mean HDL and LDL AUCs were not different between groups (data not shown). The mean E2 levels were higher in the female (25±2 pg/mL) as compared with the male (18±1 pg/mL) rabbits (P<0.05). The mean numbers of adherent monocytes per 13.5 mm2 in females and males were 0 and 1.4±1.2 monocytes per 13.5 mm2, respectively, and were not significantly different from one another.
The mean cholesterol AUCs for gonad-intact female and male
rabbits after 4 weeks of HC were not significantly different from one
another throughout the feeding period (P>0.05) (Figure 2). Mean HDL and LDL AUCs were not
different between groups (data not shown). The mean number of monocytes
adherent to the endothelial surface and the number that
migrated subendothelially were significantly lower in
the female as compared with the male animals (Figure 3), and the mean E2
levels were higher in the female as compared with the male animals
(Figure 3), indicating an inverse relationship between
E2 levels and monocyte adhesion and
subendothelial migration in
hypercholesterolemic animals.
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Protocol 2: Effects of E2 Supplementation on Monocyte
Adhesion in Ovariectomized Animals Fed a Cholesterol-Enriched
Diet
The mean cholesterol AUCs throughout the feeding
period for animals supplemented with OVX/placebo and
OVX/E2 were not significantly different from one
another (P>0.05) (Figure 2
). Mean HDL and LDL
AUCs were also not different between groups (data not shown). The mean
number of monocytes adherent to the endothelial surface
and the number that migrated subendothelially were
significantly greater in the placebo-supplemented than in the
E2-supplemented animals (Figure 4), and the mean E2
levels were significantly lower in the placebo-supplemented than in the
E2-supplemented animals (Figure 4), again
demonstrating an inverse relationship between E2
levels and monocyte adhesion and subendothelial
migration in animals fed a cholesterol-enriched diet.
Representative photomicrographs depicting this
relationship are shown in Figure 1.
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Protocol 3: Effects of E2 Supplementation on VCAM-1
Protein Expression in Ovariectomized Animals Fed a
Cholesterol-Enriched Diet
OVX/placebo-supplemented animals placed on HC demonstrated an
increase in VCAM-1 protein expression as compared with that seen in
OVX/placebo-supplemented animals placed on NC. In all animals, this
increase was attenuated by supplementation with
E2. In Figure 5 is
shown a Western blot from representative animals in
each group, and in Figure 6 is a
scatterplot of the arbitrary densitometric units of data from 5
individual animals in the cholesterol-fed groups that
received either placebo or E2 when the Western
blot was run on the same gel. In some experiments, we compared the
expression of VCAM-1 with GAPDH. We consistently observed a
decrease in VCAM-1 expression in E2-supplemented
animals with no change in GAPDH expression (data not shown).
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Protocol 4: Effects of E2 Supplementation on VCAM-1
Protein Expression in Cultured Endothelial Cells
Stimulated With Lyso-PC
Lyso-PC at 100 µmol/L increased the cellular content of
VCAM-1 protein by 248% as compared with vehicle (data not shown).
E2 inhibited this increase in a
concentration-dependent fashion (Figure 7). Figure 8 shows the
representative traces of FACS analysis from 2
separate experiments. ICI-182780 abolished the effects of
E2.
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, Cells were treated with Lyso-PC (100
µmol/L) for 15 hours; 
, single dose E2 1000 pg/mL for
48 hours followed by stimulation with Lyso-PC (100 µmol/L) for
15 hours; 
, single dose E2 3000 pg/mL for 48 hours
followed by stimulation with Lyso-PC (100 µmol/L) for 15 hours;
, ICI-182780 and then E2 3000 pg/mL for 48 hours
followed by stimulation with Lyso-PC (100 µmol/L) for 15 hours.
Data depict VCAM-1 induction in the presence of E2 alone or
in combination with the estrogen receptor antagonist ICI-182780 and are
expressed as a percentage of the induction achieved with Lyso-PC alone.
Data are expressed as mean±SEM of 5 separate experiments.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanisms by which females are protected from the development of atherosclerosis compared with males and the role that estrogen plays in this process are not known. The primary objective of this study was to assess whether there was a gender difference in the adhesion of monocytes to endothelial cells in vivo in animals fed a cholesterol-enriched diet and whether this can be explained on the basis of circulating E2.
Our studies demonstrate in vivo that adhesion of monocytes to endothelial cells is retarded in females as compared with males. This difference was not mediated by any changes in circulating lipids, as the male and female animals used for the study had comparable lipid levels. Thus, reduced monocyte adhesion in females may be a partial explanation for the gender differences in the extent of atherosclerosis previously reported in this species.11 12
To elucidate whether the higher E2 levels seen in the ovary-intact females accounted for the gender difference in monocyte adhesion in vivo, we studied this phenomenon in ovariectomized rabbits either supplemented with placebo or supplemented with 1.5-mg, 60-day release E2 pellets. These pellets provide near-physiological concentrations of E2, which are concentrations comparable with those seen in ovary-intact rabbits. In ovariectomized rabbits supplemented with E2, there were both significantly fewer monocytes adherent to endothelial cells and fewer subendothelial monocytes when compared with ovariectomized animals supplemented with placebo at 4 weeks of hypercholesterolemia. Again, this action of E2 was not due to any E2-induced changes in circulating lipids. It is interesting to note that the number of monocytes adherent to the endothelium in ovariectomized animals supplemented with placebo was significantly higher as compared with gonad-intact males. The role of circulating androgens on monocyte endothelial interactions was not addressed in this study. However, it is possible that androgens such as testosterone are converted into E2 by the aromatase activity present in endothelial cells.23
Products of lipid oxidation can activate the endothelium to support a specific increase in the adhesion of monocytes and T lymphocytes, but not polymorphonuclear leukocytes.13 24 25 VCAM-1 is likely to be the adhesion molecule responsible for providing this specificity.26 27 28 VCAM-1 is a member of the IgG superfamily29 and interacts with its counterreceptor, the very late appearing antigen 4 (VLA-4), a ß1-integrin expressed on the surface of monocytes and lymphocytes but not on polymorphonuclear leukocytes.30 31 In fatty streaks of rabbits with diet-induced or genetically determined hypercholesterolemia, the endothelial cells express VCAM-1 focally.32 Furthermore, the expression of VCAM-1 occurs in lesion-prone areas of the rabbit aorta as early as 1 week after initiation of a hypercholesterolemic diet before intimal macrophages accumulate.32 Our results demonstrated that E2 administration at physiological concentrations to ovariectomized rabbits fed a cholesterol-enriched diet markedly decreased VCAM-1 expression when compared with ovariectomized rabbits implanted with a placebo pellet. We did not specifically evaluate whether the VCAM-1 was expressed predominantly in the endothelial cells or vascular smooth muscles. However, other investigators33 have reported that in rabbits fed a cholesterol-enriched diet for 6 weeks or less, VCAM-1 was expressed predominantly in endothelial cells. It is therefore likely that the differences in VCAM-1 expression observed in our studies were due to differences in endothelial cell VCAM-1 expression. Results from our studies do not indicate whether the effects of E2 in decreasing VCAM-1 expression in vivo are a result of its antioxidant properties34 35 or whether this action is mediated by the binding of E2 to its receptor in endothelial cells.
To corroborate our in vivo data and to try and determine the mechanism by which E2 regulates VCAM-1 expression, we also investigated whether there was a corresponding effect of E2 in the presence of an estrogen receptor antagonist on VCAM-1 protein expression by RAECs in vitro. We used lyso-PC to activate our endothelial cells, as lyso-PC is a major chemotactic lipid component of both oxidized LDL and ß-VLDL.36 37 In addition, the concentration of lyso-PC is increased in atherosclerotic arterial lesions in animals fed an atherogenic diet38 and has been demonstrated to be a selective chemoattractant for mononuclear leukocytes.36 Lyso-PC has also been shown to selectively induce both VCAM-1 and intracellular adhesion molecule-1 in RAECs.39 In keeping with previous studies,39 we also observed that lyso-PC (100 µmol/L) induced the expression of VCAM-1 on endothelial cells. Our results further indicated that E2 at concentrations of 1000 and 3000 pg/mL reduced the surface expression of VCAM-1 on endothelial cells in culture in the presence of lyso-PC. This action of E2 in attenuating VCAM-1 expression in vitro was abolished in the presence of the E2 antagonist, ICI-182780, which blocks the interaction of E2 with the estrogen receptor.16 This suggests that the action of E2 in regulating VCAM-1 expression was at least in part due to the interaction of E2 with its receptor and not via an indirect antioxidant effect of E2.
The action of E2 in modulating VCAM-1
expression in endothelial cells has also been studied
by other investigators, but only in vitro, and the results are
controversial. In studies of human umbilical vein
endothelial cells (HUVECs), E2
suppressed the induction of VCAM-1, E-selectin, and intracellular
adhesion molecule-1 mRNA expression by
interleukin-1ß.40 41 In contrast, Cid et
al42 observed that E2 did not affect
basal VCAM-1 expression but caused a 30% to 50% increase in the
presence of tumor necrosis factor (TNF) and had no effect when the
HUVECs were stimulated with IL-1. It has also been reported that
E2 increased the TNF-
–induced expression of
E-selectin in endothelial cells in culture but had no
effect on the expression of VCAM-1.43 These contrasting
observations may in part be due to differences in the cell types used
(HUVECs versus RAECs) and the degree to which they express estrogen
receptors, differences in the concentrations of
E2 administered (ranging from 50 to 5000 pg/mL),
and differences in the agents used to induce VCAM-1 expression
(lyso-PC, IL-1ß, and TNF-).
In this regard, in both the present study and in those previously reported, the concentrations of E2 required for inhibition of VCAM-1 expression in vitro were in the supraphysiological range. This is in sharp contrast to the E2-mediated inhibition of VCAM-1 expression in vivo that occurred at physiological E2 concentrations. At present, it is unclear why there is this discrepancy between the concentration of E2 required for the suppression of VCAM-1 expression in vivo versus in vitro. It is possible that in vivo, E2 acts by the following 2 mechanisms: inhibition of LDL oxidation with a concomitant decrease in the amount of lyso-PC formed, and a direct estrogen receptor-mediated action on the endothelial cells. Estrogen receptors are present in both endothelial cells and vascular smooth muscles in vivo.44 45 46 47 However, the relative degree to which estrogen receptors are expressed by these cells under in vitro conditions has not yet been reported. A higher expression of estrogen receptors in vivo could partly explain why lower concentrations of circulating E2 inhibit VCAM-1 expression in vivo relative to the much higher concentrations of E2 required in vitro. It is also possible that E2 may be converted into a more active metabolite in vivo, which may also partially account for the higher concentrations of E2 required to suppress VCAM-1 expression in vitro. Further work is in progress to assess this aspect.
Finally, our data also demonstrated that E2 administration reduced the number of monocytes that migrated into the subendothelial space. This could simply be a result of the reduction in the number of adhesion sites. However, this may also be due to an E2-mediated inhibition of expression of monocyte-specific chemokines. For example, in preliminary studies of a similar group of hypercholesterolemic rabbits, we observed that E2 inhibits expression of the monocyte chemotactic protein-1 (MCP-1).48 Thus, estrogen appears to have the combined property of inhibiting both monocyte adhesion and the subsequent recruitment of monocytes into the arterial intima.
Monocyte adhesion and subendothelial migration are key cellular events generally associated with the initiation of the atherogenic process. In this regard, an estrogen-mediated inhibition of these processes could not account for the fact that the protection against CVD seen in women does not manifest itself until middle age, when inflammatory cell–rich fatty streaks have generally progressed to more advanced atherosclerotic lesions. However, monocyte recruitment is part of a fibroproliferative inflammatory response that is continuous throughout the entire atherogenic process. For example, VCAM-1 expression with an associated leukocyte infiltration has been demonstrated in microvessels within advanced human plaques.49 Furthermore, there is now ample evidence that advanced plaques most frequently rupture at sites enriched with inflammatory cells and lipid.50 51 Plaque rupture in turn leads to the formation of occlusive thrombosis and the clinical sequelae of CVD. Thus, the effect of estrogen in inhibiting VCAM-1 and MCP-1 expression and monocyte adhesion and subendothelial migration is likely to have a profound impact on all stages in the atherogenic process and may also account for the protective effects in women before menopause.
In conclusion, our results suggest that reduced inflammatory cell infiltration into the artery wall due to an estrogen-mediated inhibition of the expression of VCAM-1 and/or MCP-148 is in part responsible for the protection from atherosclerosis that is seen in women.
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Acknowledgments |
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This work was supported in part by the American Federation for Aging Research, Inc; the Hartford Foundation; National Heart, Lung, and Blood Institute Grants HL-46843 and HD-31467, and the Laubisch Fund for Cardiovascular Research. We thank Janis Cuevas for her technical expertise in the animal preparations and Jerry Ricks for his technical expertise in the scanning electron microscopy.
Received May 5, 1999; accepted June 11, 1999.
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