© 1999 American Heart Association, Inc.
Original Contributions |
Modified LDL–Mediated Increases in Endothelial Layer Permeability Are Attenuated With 17ß-Estradiol
From the Division of Cardiovascular Medicine, University of California, Davis (G.G., K.A.R., A.E.M., J.C.R.), and The Scripps Research Institute (C.L.B.), La Jolla, Calif.
Correspondence to John C. Rutledge, MD, Division of Cardiovascular Medicine, One Sheilds Drive, TB 172, University of California, Davis, CA 95616. E-mail jcrutledge{at}ucdavis.edu
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Abstract—Current research suggests that estrogen may have primary effects on the artery wall. To investigate the mechanisms of female sex hormone actions in the artery wall, we used an isolated, perfused, rat carotid artery model to examine the effects of estradiol on the rates of accumulation of normal (N-LDL) and minimally modified (MM-LDL) low density lipoprotein in ovariectomized rats. N-LDL, MM-LDL, and oxidized LDL (OX-LDL) were fluorescently labeled and perfused into individual arteries. The rate of LDL accumulation was measured by quantitative fluorescence microscopy before and after treatment with estradiol (1 nmol/L, 272 pg/mL). Estradiol had no effect on the rate of N-LDL accumulation (45±12 versus 48±15 ng cholesterol per cm2 per h). However, estradiol significantly decreased the rate of MM-LDL (240±48 versus 160±48 ng cholesterol per cm2 per h; P<0.05) and OX-LDL (191±53 versus 112±36 ng cholesterol per cm2 per h; P<0.05) accumulation. Further experiments showed that perfusion of unlabeled MM-LDL (100 µg/mL) increased endothelial layer permeability when the rate of accumulation of a water-soluble, fluorescently labeled, reference molecule (64 000–molecular weight dextran) was determined before and after perfusion of MM-LDL (319±96 versus 510±191 ng per cm2 per h, n=6 arteries; P<0.05). Estradiol prevented the expected increase in the rate of dextran accumulation when perfused with MM-LDL (control, 415±49 ng per cm2 per h and MM-LDL+estradiol, 415±160 ng per cm2 per h). Our studies show that estradiol prevents compromise of the endothelial barrier mediated by MM-LDL and attenuates accumulation of MM-LDL in the artery wall.
Key Words: artery • arteriosclerosis • LDL • estrogen • oxidized LDL • endothelium • permeability
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Introduction |
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Acardinal characteristic of the development of atherosclerosis is the accumulation of LDL within the arterial wall.1 2 3 Sites of relative increase in LDL permeability have been identified in normal arteries4 and can be induced by injury to the endothelium.5 6 7 8 9 Alternatively, others have reported increased binding and decreased efflux of LDL from the vascular wall, even under conditions where LDL influx was not affected.10 11 Therefore, LDL accumulation in the artery wall potentially occurs by several mechanisms, perhaps depending on the stage of atheroma formation.
A large body of evidence indicates that modification of LDL in the artery wall promotes atherosclerosis.12 Recent studies demonstrate evidence of modification of LDL in the blood.13 14 15 16 17 Furthermore, a previous study showed increased accumulation and degradation of oxidized LDL (OX-LDL) in the artery wall in vivo.18 These studies suggest that the reason there is increased accumulation and degradation of OX-LDL is because arterial permeability to undegraded, OX-LDL is greater than arterial permeability to native LDL (N-LDL). However, the precise mechanism(s) by which modified LDL (MM-LDL) affects LDL flux in the artery wall, such as increases in permeability to LDL, remains incompletely understood.
Modification of LDL (eg, oxidation), either in the circulation or the artery wall, could promote atherosclerosis by increasing LDL accumulation in the artery. Also, alteration of constituents of the artery wall, such as proteoglycans, could influence LDL binding to the artery wall. Furthermore, oxidant-mediated injury of endothelial cell membranes could compromise the endothelial layer barrier to macromolecules in arteries.5 To counteract oxidant stress effects on LDL and/or the artery wall, a number of potential antioxidants have been examined. In particular, some estrogens (eg, 17ß-estradiol) are known to reduce LDL accumulation in the artery wall19 20 and to be potent antioxidants.21 22 23 In addition, estrogens may protect endothelial cells from oxidative damage.21 22 This action would be expected to prevent increased LDL permeation into the artery wall. Furthermore, estradiol has been shown to inhibit LDL oxidation in vivo at supraphysiological24 and physiological25 concentrations. These data suggest that some estrogens may beneficially affect LDL or the artery wall by their antioxidant effects.
To examine the interactions of MM-LDL and estradiol in the artery wall, we developed a system to perfuse individual arteries with N-LDL, MM-LDL, and OX-LDL and to test the actions of estradiol on LDL flux in the artery wall. Our results show greater accumulation of MM-LDL than N-LDL. Furthermore, estradiol decreased accumulation of both MM-LDL and OX-LDL and prevented compromise of endothelial layer integrity induced by MM-LDL.
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Methods |
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Chemicals and Materials
Krebs-Henseleit buffer consisted of (in mmol/L) NaCl 116, KCl 5, CaCl2 1.2, NH2PO4 1.2, and glucose 11. 17ß-Estradiol (No. 125H1081) and EDTA were obtained from Sigma Chemical Co. Low-endotoxin Dulbecco's modified Eagle's medium was prepared in The Scripps Research Institute media kitchen.
Experimental Animals
Female Crl:CD® (SD) BR rats (100 to 150 g) obtained from
Charles River Breeding Laboratories (Boston, Ma) were used in
all experiments. Animals were individually housed in plastic cages with
a stainless steel cover. They were given tap water and Purina
Laboratory Rodent Chow ad libitum. The animal housing facility and
experimental protocol was approved by the Animal Use Committee at the
University of California at Davis.
Ovariectomy Surgery
Rats were anesthetized with an
intraperitoneal injection of ketamine (80
mg/kg) and xylazine (10 mg/kg). A 0.5- to 1.0-cm longitudinal incision
was made in the midline dorsal area of the back from the second to the
fifth lumbar vertebrae.26 A small peritoneal incision was
made and the ovaries were located. Once the ovaries were tied off at
the tip of the uterus, they were removed. Vexaband tissue glue and
sutures (absorbable 3-0, Dexon II) were used to close the incisions.
Animals were given an intramuscular injection of an antibiotic
(enrofloxicine, 10 mg/kg) and placed on a heating pad until they
recovered.
Isolation and Modification of LDLs
Human blood was collected in citrate from healthy male donors at
the San Diego Blood Bank. LDL was isolated by density gradient
ultracentrifugation and was stored in Dulbecco's
modified Eagle's medium with 0.3 mmol/L EDTA.27
Immediately before oxidation, antioxidants were removed from LDL by
overnight dialysis against saline. Lipoproteins were modified by
incubating LDL (2.5 mg protein per mL) with 5 µmol/L copper
acetate (Cu2+), and the extent of modification
was measured by thiobarbiturate-reactive substances
(TBARS).27 28 MM-LDL (TBARS=2.4 to 6.8 nmol
malondialdehyde equivalent s per mg protein) was obtained by incubating
human N-LDL with Cu2+ for 1 hour, and the OX-LDL
(TBARS=7.5 to 32.7 nmol/mg) was obtained by incubating N-LDL with
Cu2+ for 18 hours. In comparison, N-LDL had TBARS
values of 0.00 to 1.71 nmol malondialdehyde equivalents per mg protein.
N-LDL, MM-LDL, and OX-LDL used in a given protocol were from the same
male donor. Each of the protocols was conducted with LDL from a
different donor.
Fluorescent Labeling of LDL and Dextran
N-LDL, MM-LDL, and OX-LDL were labeled with the
fluorescent hydrocarbon probe
1,1-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine
(DiI) as described by Pitas et al.29 Dil exchanges with
lipoprotein phospholipid and does not transfer the fluorescent
probe because the 2 octadecyl moieties make the compound hydrophobic.
The spectral properties of Dil are the following: excitation maximum at
540 nm and emission maximum at 556 nm. Dextran (64 000 molecular
weight) was obtained from Sigma labeled with TRITC. The spectral
properties of TRITC are excitation maximum at 554 nm and emission
maximum at 573 nm.
Experimental Protocol
Four to 6 weeks after ovariectomy, the arterial
perfusion experiments were performed.6 30 Rats were
anesthetized by intraperitoneal injection
of sodium pentobarbital (35 mg/kg). After the rat was
anesthetized, a midline incision was made in the neck, and both
carotid arteries were dissected free from connective tissue. An
incision was made in the proximal segment of the artery, and a cannula
(23-gauge tubing adapter connected to polyethylene-50 tubing)
was inserted into the carotid artery. The cannula was tied in place
(silk 6-0). Another incision was made in the distal portion of the
artery proximal to the bifurcation of the internal and external carotid
arteries. Another cannula (identical to the proximal cannula) was
inserted into the distal carotid artery and tied in place. The artery
was removed from the animal and placed into a fluid-filled, clear
Plexiglas chamber on the microscope stage (perfusion chamber).
Throughout the surgery, the artery was superfused with Krebs-Henseleit
solution gassed with 95% O2 and 5%
CO2. The Krebs-Henseleit solution traveled from a
reservoir through a heating coil to the superfusate chamber and
was maintained at 37°C throughout the experiment.
To reduce the number of animals needed for these experiments, both carotid arteries were isolated and used for perfusion. A single experiment was performed on each artery, and protocols were performed in sequence. For example, in protocol 1 the rates of LDL accumulation for N-LDL, MM-LDL, and OX-LDL were performed in 3 sequential arteries (2 carotid arteries from 1 animal and 1 carotid artery from the second animal). The sequence began again with the second carotid artery from the second animal.
The artery was perfused with Krebs-Henseleit solution containing 1% BSA (perfusate solution), also gassed with 95% O2 and 5% CO2. The perfusate solution entered the artery through the proximal cannula and exited the artery through the distal cannula. By using 3-way stopcocks and 2 parallel sets of tubing, the artery was perfused with either the nonfluorescent solution or the fluorescent solution. The arteries were perfused antegradely by a peristaltic pump. A normal flow rate was maintained in the carotid artery (7 mL/min). Periodic measurements of pH and partial pressures of O2 and CO2 were obtained to maintain physiological conditions.
Experimental Apparatus
The experimental apparatus used to measure LDL flux
consisted of an inverted Nikon microscope with objective (planx6.3;
numerical aperture, 0.2) mounted on the microscope head. The
fluorescence image was transmitted through a beam splitter to a
Dage low-light television camera and a Nikon P101 photometer. The
photometer used to measure changes in fluorescence intensity
was connected to a chart recorder and computer. Output from the
camera was input into the videocassette recorder and to a
high-resolution monitor. Images of the labeled LDL flux were
recorded on videotape and assessed later by image
analysis.31
Measurement of LDL Accumulation and Lumen Volume
Several measurements were made during and after perfusion of
each carotid artery with fluorescently labeled molecules.
Initially, a baseline measurement of fluorescence intensity
(If) was established while the artery was perfused with the
nonfluorescent solution (Figure 1
). Then, the artery was perfused with
the fluorescent solution, and a step increase in
fluorescence intensity was observed. Fluorescence
intensity rapidly increased to approach an asymptotic value
(If0). Simultaneously, LDL may become
associated with the luminal surface of the artery or enter the artery
wall. If the perfusate concentration of the
fluorescently labeled molecules and perfusate flow
remain constant, then If0 determined at the
asymptotic value estimated the lumen volume.30 As the
artery continued to be perfused with fluorescent solution, some
of the labeled molecules became bound to the
endothelium and/or crossed into the artery wall. After
washout with the nonfluorescent solution, labeled molecules
that remained on the luminal surface or in the artery wall were termed
If accumulation. Therefore, molecule accumulation in the artery wall
was determined photometrically (in millivolts) and was a measure of the
number of fluorescent molecules that remained on the lumen
surface or in the artery wall.6 The rate of molecule
accumulation is If accumulation divided by perfusion time. Using this
approach, we were able to measure If accumulation multiple times
(Figure 2). Furthermore, the effect of
estradiol can be determined by adding estradiol to both the
fluorescent and nonfluorescent solutions after control
rates of If accumulation have been determined.
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Indicates initiation of
perfusion of the fluorescently labeled LDL solution.
Then, the artery was perfused with the nonfluorescent buffer
solution again. Little or no fluorescent N-LDL remained in the
artery wall after the washout with the nonfluorescent solution.
For MM-LDL during perfusion with the solution containing
fluorescently labeled MM-LDL, increased fluorescence
intensity was observed with time, indicating increased accumulation of
MM-LDL on or in the artery wall. After washout of the artery with the
nonfluorescent solution, a residual increase in
fluorescence intensity above baseline was observed (If
accumulation). This represents the MM-LDL that accumulated in
the artery wall and on the intimal surface over the time of perfusion
with the fluorescently labeled MM-LDL.
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To relate fluorescence intensity (If) to lumen volume, we
determined the fluorescence intensity of a given amount of LDL
in the artery lumen. The volume of the artery was determined by
· radius2 · length. Thus,
fluorescence intensity in the lumen can be related to units of
volume to indirectly measure artery lumen volume.
Accumulation (If) in millivolts was converted to nanograms of
cholesterol per square centimeter per hour as we have
described previously.32 In summary, we assume the
relationship
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The rates of DiI–N-LDL, DiI–MM-LDL, and DiI–OX-LDL efflux were determined, as we have described previously.6 In summary, the rate of decay of fluorescence intensity was determined after the fluorescently labeled LDL solution was washed from the vessel lumen. The decay in fluorescence then represents the efflux of LDL back into the artery lumen.
Plasma Estradiol Measurements
After isolation and cannulation of the carotid artery, blood was
collected from each animal via the right atrium by using a 22-gauge
needle and heparinized syringe. Blood was transferred to evacuated
containers and centrifuged (2800 rpm for 10 minutes). Plasma
was immediately separated from red blood cells and kept on ice. The
samples were sent to the Endocrinology Laboratory of the School of
Veterinary Medicine at the University of California at Davis for
analysis of estradiol concentration by using a charcoal-based,
tritium-labeled radioimmunoassay.33 The mean plasma
estradiol level 4 to 6 weeks after ovariectomy was 11.2±1.4 pg/mL.
Statistical Analysis
Multiple measurements of the rate of LDL accumulation and lumen
volume were made from each artery for each treatment. To give each
artery and each treatment equal weight, the mean of all measurements of
the rates of LDL accumulation and lumen volume were determined for each
treatment in each artery. Group data of each treatment are
presented as the mean±SEM. Group data were analyzed by
repeated-measures ANOVA (Sigma Stat statistical hardware, Jandel Corp).
Significant effects were assessed with the Student-Newman-Keuls
multiple-comparison test. A probability level of P
0.05 was
considered statistically significant.
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Results |
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Accumulation of N-LDL, MM-LDL, and OX-LDL in the Artery Wall: Protocol 1
N-LDL, MM-LDL, and OX-LDL were labeled with DiI. Individual arteries were perfused with DiI–N-LDL (4 arteries), DiI–MM-LDL (4 arteries), or DiI–OX-LDL (4 arteries). The concentration of all fluorescently labeled LDL solutions was 0.042 mg/mL protein and 0.083 mg/mL cholesterol. In each artery, a baseline level of fluorescence intensity was determined by perfusing the artery with the nonfluorescent buffer solution (Figure 1
). Then, fluorescently labeled LDL was perfused for 10
minutes, followed by a 10-minute perfusion with nonfluorescent
perfusate. The rate of LDL accumulation was determined after
each 10-minute perfusion with fluorescently labeled LDL. Each
artery had the rate of LDL accumulation determined 5 times (Figure 2). There was a significant increase in the rate of LDL
accumulation during perfusion with MM-LDL and OX-LDL (175±32
and 178±5 ng cholesterol per cm2 per
h, respectively) compared with N-LDL (64±16 ng cholesterol
per cm2 per h; P<0.001; Figures 1, 3, and 4 and
Table, protocol 1). Increased MM-LDL and
OX-LDL accumulation was apparent with the first perfusion of MM-LDL and
OX-LDL and remained elevated for the duration of the experiment. No
trend was noted toward increased MM-LDL or OX-LDL accumulation with
time.
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Effect of Estradiol on N-LDL, MM-LDL, and OX-LDL Flux in the Artery
Wall: Protocol 2
Fluorescently labeled N-LDL, MM-LDL, or OX-LDL was
perfused into separate arteries (N-LDL, n=8 arteries; MM-LDL, n=10
arteries; or OX-LDL, n=10 arteries) in the same concentrations as
described above. Two arteries (N-LDL) developed leaks and were
therefore technically unacceptable. In each artery, the rate of N-LDL,
MM-LDL, or OX-LDL accumulation was determined. Then, while the
nonfluorescent Krebs-Henseleit solution was being perfused,
estradiol (1 nmol/L, 272 pg/mL) was added to both the
nonfluorescent and fluorescent perfusion solutions
(Figure 5). Estradiol was solubilized in
ethanol and diluted in Krebs-Henseleit buffer to its final
concentration (0.000020% ethanol by volume). This concentration of
estradiol has been shown not to affect the rate of LDL accumulation in
the artery wall in short-term studies.32
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After estradiol was added to both the nonfluorescent and the
fluorescently labeled LDL solutions, the artery was perfused
for 10 minutes with the nonfluorescent solution. Then, the
artery was perfused with the fluorescently labeled LDL solution
for 10 minutes. Therefore, each artery was treated with estradiol for
20 minutes before the first measurements of the rates of accumulation
of N-LDL, MM-LDL, or OX-LDL were made. As in the previous protocol, the
rates of OX-LDL and MM-LDL accumulation were significantly greater than
that of N-LDL accumulation. Furthermore, the rate of MM-LDL
accumulation tended to be greater than that of OX-LDL accumulation. The
rate of N-LDL accumulation (45±12 ng per cm2 per
h) was not changed after estradiol was added (48±15 ng per
cm2 per h; Figure 6). There was, however, a significant
decrease in MM-LDL accumulation after the addition of estradiol to the
perfusate (240±48 versus 160±48 ng per
cm2 per h; P<0.05). The rate of
accumulation of OX-LDL was 191±53 ng per cm2 per
h. Addition of estradiol significantly decreased OX-LDL accumulation
(112±36 ng per cm2 per h;
P<0.05).
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The rates of DiI–N-LDL, DiI–MM-LDL, and DiI–OX-LDL efflux were determined in these same arteries. The rates of efflux before and after addition of estradiol to the perfusate were as follows: N-LDL, 139±33.6 and 175±55 ng per cm2 per h; MM-LDL, 28.6±7.6 and 31±10 ng per cm2 per h; and OX-LDL, 33±9 and 44±14 ng per cm2 per. The rate of N-LDL efflux was significantly greater than that of MM-LDL or OX-LDL. No differences in rates of efflux were noted before and after addition of estradiol to perfusions containing N-LDL, MM-LDL, and OX-LDL.
Estradiol did not significantly change lumen volume when the artery was
treated with N-LDL, MM-LDL, or OX-LDL. For example, when the
concentration of the N-LDL solution was 0.042 mg protein per mL, the
volume of the artery contained within the measuring window was 9.4
µL, and the fluorescence intensity was 57 mV when the vessel
lumen volume was filled with DiI–N-LDL, then each millivolt
represented 
0.17 µL. Therefore, during perfusion with
N-LDL, estradiol increased the lumen volume from 9.4 µL to only 10.2
µL.
Effects of MM-LDL on Endothelial Layer
Permeability: Protocol 3
To determine the effect of MM-LDL on endothelial
layer permeability, we perfused the arteries with
nonfluorescently labeled MM-LDL at a concentration range of 10
to 350 µg/mL protein. Endothelial layer permeability
after perfusion with MM-LDL was determined by measurement of
accumulation of a nonlipid, water-soluble reference molecule (dextran,
64 000 molecular weight, at 75 µg/mL and labeled with TRITC).
Significant increases in the rates of dextran accumulation were first
detected after perfusion with 100 µg/mL MM-LDL (319±96 versus
510±191 ng per cm2 per h; n=6 arteries;
P<0.05). As in an in vivo control, we conducted a
whole-animal perfusion and showed that MM-LDL did, in fact, accumulate
in the carotid artery (Figure 7).
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To compare the effect of MM-LDL on endothelial layer permeability with other lipoproteins, we perfused arteries with nonfluorescently labeled HDL (0.2 mg/mL protein, 0.16 mg/mL cholesterol). No increase in the rate of dextran accumulation was noted after the artery wall was perfused with HDL (447 versus 383 ng cholesterol per cm2 per h; n=2 arteries).
Impact of Estradiol on MM-LDL–Mediated Dextran Accumulation:
Protocol 4
Next, we determined the effect of treatment of the artery with
estradiol on the rate of dextran accumulation before and after
perfusion with MM-LDL (100 µg/mL). In each artery (n=6), the rate of
dextran accumulation was determined during perfusion of the solution
containing TRITC-labeled dextran (415±49 ng per
cm2 per h). Then, estradiol (272 pg/mL) was added
to both the fluorescent and nonfluorescent solutions,
and repeated measurements of the rate of dextran accumulation were made
(479±85 ng per cm2 per h). The rate of dextran
accumulation after estradiol was added to the perfusate was not
significantly different from control measurements. Then, MM-LDL (100
µg/mL) was added to the nonfluorescent solution, and repeated
measurements of the rate of dextran accumulation after MM-LDL exposure
were made in the presence of estradiol (415±160 ng per
cm2 per h). Treatment of the artery with
estradiol prevented the expected increase in the rate of dextran
accumulation when perfused with MM-LDL.
Thus, treatment with estradiol prevented MM-LDL–mediated compromise of
the endothelial barrier function.
To determine the effects of N-LDL on estradiol interactions with MM-LDL, we performed additional control experiments. First, we measured the rate of dextran accumulation (240±18.2 ng per cm2 per h) in the presence of N-LDL (100 µg/mL). Then, we added MM-LDL (100 µg/mL) to the perfusate and again measured the rate of dextran accumulation with N-LDL in the perfusate (318±17.3 ng per cm2 per h; a 32.5% increase in the rate of dextran accumulation). The other carotid artery from the same animal also was used. Initially, we measured the rate of dextran accumulation in the presence of N-LDL and estradiol (at the same concentrations; 324±3.6 ng per cm2 per h). Next, MM-LDL was added to the perfusate, and the rate of dextran accumulation was determined in the presence of N-LDL and estradiol (348±81 ng per cm2 per h; a 7.4% increase in dextran accumulation). These experiments also showed that MM-LDL increased endothelial layer permeability and that estradiol reduced compromise of the endothelial layer in the presence of N-LDL.
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Our study showed that estradiol attenuated the accumulation of MM-LDL in the walls of isolated, perfused, rat carotid arteries. Accumulation of MM-LDL and OX-LDL decreased when estradiol was added to the perfusate. Perfusion of MM-LDL increased the accumulation of fluorescently labeled dextran, a marker of increased endothelial layer permeability. The effect of MM-LDL on endothelial layer permeability was nullified by addition of estradiol to the perfusate and indicates that the protective effects of estradiol on the artery wall are mediated, in part, by attenuation of MM-LDL–induced endothelial cell injury.
The interactions of ovarian sex hormones with the artery wall are complex and multifaceted. Physiological concentrations of estrogens have been shown to act directly on the artery wall,32 34 to attenuate atherosclerosis,35 and to reduce morbidity and mortality associated with atherosclerotic cardiovascular disease in postmenopausal women.36 In comparison, supraphysiolgical concentrations of some estrogens (for example, 17ß-estradiol) can increase LDL accumulation in the artery wall.32 However, the mechanisms by which estrogens affect the interaction of MM-LDL with the artery wall are incompletely understood and were, therefore, the subject of this article.
To study the interactions of MM-LDL with the artery wall, we used the most direct method possible, by perfusing MM-LDL into the lumen of the artery. This approach is advantageous because it allowed us to directly perfuse a known amount of modified LDL into the artery and analyze the interactions of MM-LDL with the artery wall. This approach enabled us to precisely manipulate the compositions of the perfusate and superfusate solutions and carefully control arterial flow and hydrostatic pressure, hemodynamic parameters known to influence LDL flux into the artery wall.7 8 37 The approach that we have taken enabled us to study the mechanisms of LDL flux into the artery wall and is a bridge between previous in vitro and cell culture experiments and experiments performed on whole-animal preparations.5 18 38
The literature suggests that LDL is primarily modified in the arterial wall because of the large antioxidant capacity of the blood.39 However, recent data show that modification of LDL can occur in the blood.13 14 15 16 17 Furthermore, prolonged LDL circulation times in the blood increase LDL's propensity to oxidation and may lead to LDL modification before it enters the artery wall.40 Previous work from this laboratory showed that even short exposures to environmental tobacco smoke (4 hours) increased LDL accumulation in the artery wall. This effect appears to be related to modification of LDL by the products of environmental tobacco smoke, a process that may occur in blood.30
We saw a tendency for MM-LDL to accumulate in the artery wall to a
greater extent than did OX-LDL. This could result from a number of
possible mechanisms, including induction of substances that increase
endothelial layer permeability, such as
cytokines (eg, tumor necrosis factor-
). Also, this
interaction could be related to greater oxidative proteolysis of apoB
during a longer duration of copper-mediated oxidation of LDL. Previous
studies showed high-avidity binding of constituents of apoB (eg,
apoB17) with matrix molecules of the artery wall, such as heparan
sulfate proteoglycans.41 Extensive oxidation of LDL (ie,
OX-LDL) may eliminate the exposed portions of apoB on
LDL.27 These studies imply that constituents of apoB are
important or necessary for the interaction of LDL with the artery
wall.
Researchers from a number of laboratories have investigated the
potential antioxidant effects of estrogens. Maziere et
al21 found that 17ß-estradiol, estriol, and estrone
inhibited LDL oxidation. Also, pretreatment of
endothelial cells and monocyte-like cells with
estradiol 24 hours before exposure to OX-LDL prevented cytotoxic
effects associated with Ox-LDL. In a similar study, Negre-Salvayre et
al22 reported that estradiol protected cultured bovine
endothelial cells from cytotoxic effects of MM-LDL.
However, testosterone, progesterone, and cholesterol had no
protective effects. Huber et al42 and Maziere et
al21 reported that estrogens inhibited LDL oxidation and
the accumulation of cholesteryl esters in macrophages.
Furthermore, in perfused arteries, estradiol was shown to reduce
oxidant stress mediated by tumor necrosis factor-.43
Therefore, a considerable body of data indicates significant
antioxidant effects associated with estrogens.
In our experiments, LDL was already modified when perfused into the arteries. Although estradiol could prevent further modification of LDL in the artery, a primary artery wall effect was suggested. Our studies showed that MM-LDL increased endothelial layer permeability, suggesting endothelial cell injury. Estradiol appeared to prevent this action. We speculate that estradiol partitioned into endothelial cell plasma membranes and stabilized the membranes, possibly by preventing lipid peroxidation or by altering fluidity.21 The antioxidant properties of estradiol defended endothelial cells from MM-LDL–mediated oxidant stress. Thus, estradiol stabilized the plasma membrane and maintained endothelial layer integrity.
Our studies demonstrated that not only did MM- and OX-LDL accumulate to a greater extent in the artery than did N-LDL but also that their rate of efflux was reduced. This latter effect suggests increased binding of MM- and OX -LDL once they entered the artery wall. Therefore, although increased endothelial layer permeability appeared to be the primary action of MM- and OX-LDL on the vessel wall in these studies, increased binding of MM- and OX-LDL to the artery wall also could play a role in total LDL accumulation. However, increased binding, as measured by the rate of efflux, was not prevented by estradiol, as was the increased endothelial layer permeability.
Conclusions
This study and others indicate that an important effect of
estradiol treatment is protection of the artery wall from
MM-LDL–mediated injury. Mechanistically, estradiol prevented
MM-LDL–mediated injury. Although estradiol is likely to have multiple
protective effects to prevent atherosclerosis, our
study shows that some of the actions of MM-LDL can be ameliorated with
estradiol. Because MM-LDL appears to play a critical role in the
development of atheroma, hormone replacement therapies may
mediate some of LDL's detrimental effects by the antioxidant
properties associated with estrogens.
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Acknowledgments |
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This project was supported by National Institutes of Health (Bethesda, Md) grants HL-55667 (to Dr Rutledge) and HL-50060 and HL-55517 (to Dr Banka). We wish to thank Mable Woo for her careful technical support in this project and Jeannette Peacock for her secretarial assistance.
Received October 8, 1997; accepted August 5, 1998.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Hoff HF, Jackson RL, Titus JL, De Bakey ME, Gotto Jr AM. Plasma lipoproteins and atherosclerosis in man: an immunohistochemical study. Cardiovasc Res Center Bull. 1973;12:29–39.
2. Goldstein JL, Brown MS. Lipoprotein receptors, cholesterol metabolism, and atherosclerosis. Arch Pathol. 1975;99:181–184.[Medline] [Order article via Infotrieve]
3. Heinecke JW. Mechanisms of oxidative damage of low density lipoprotein in human atherosclerosis. Curr Opin Lipidol. 1997;8:268–274.[Medline] [Order article via Infotrieve]
4.
Herrmann RA, Malinauskas RA, Truskey GA.
Characterization of sites with elevated LDL permeability at
intercostal, celiac, and iliac branches of the normal rabbit aorta.
Arterioscler Thromb. 1994;14:313–323.
5.
Rangaswamy S, Penn MS, Saidel GM, Chisolm GM.
Exogenous oxidized low-density lipoprotein injures and alters the
barrier function of endothelium in rats in vivo.
Circ Res. 1997;80:37–44.
6.
Rutledge JC, Woo MM, Rezai AA, Curtiss LK, Goldberg
IJ. Lipoprotein lipase increases lipoprotein binding to the artery wall
and increases endothelial layer permeability by
formation of lipolysis products. Circ Res. 1997;80:819–828.
7.
Nielsen LB, Nordestgaard BG, Stender S, Kjeldsen K.
Aortic permeability to LDL as a predictor of aortic
cholesterol accumulation in cholesterol-fed
rabbits. Arteriosclerosis. 1992;12:1402–1409.
8. Born GVR, Shafi S. Evidence against the atherogenic passage of low density lipoproteins between vascular endothelial cells in conscious rabbits. J Physiol. 1989;418:80.
9.
Rutledge JC, Curry FE, Blanche P, Krauss RM. Solvent
drag of LDL across mammalian endothelial barriers with
increased permeability. Am J Physiol. 1995;268:H1982–H1991.
10.
Schwenke DC, Carew TE. Initiation of atherosclerotic
lesions in cholesterol-fed rabbits, II: selective retention
of LDL vs selective increases in LDL permeability in susceptible sites
of arteries. Arteriosclerosis. 1989;9:908–918.
11. Rutledge JC, Goldberg IJ. Lipoprotein lipase (LpL) affects low density lipoprotein (LDL) flux through vascular tissue: evidence that LpL increases LDL accumulation in vascular tissue. J Lipid Res. 1994;35:1152–1160.[Abstract]
12.
Navab M, Berliner JA, Watson AD, Hama SY, Territo MC,
Lusis AJ, Shih D, Van Lenten DJ, Frank JS, Demer LL. The Ying and Yang
of oxidation in the development of the fatty streak: a review based on
the 1994 George Lyman Duff Memorial Lecture. Arterioscler Thromb
Vasc Biol. 1996;16:831–842.
13.
Demuth K, Myara I, Chappey B, Vedie B, Pech-Amsellem
MA, Haberland ME, Moatti N. Cytotoxic electronegative LDL subfraction
is present in human plasma. Arterioscler Thromb Vasc
Biol. 1996;16:773–783.
14.
Sevanian A, Hwang J, Hodis HN, Cazzolato G, Avogaro P,
Bittolo-Bon G. Contribution of an in vivo oxidized LDL to LDL oxidation
and its association with dense LDL subpopulations. Arterioscler
Thromb Vasc Biol. 1996;16:784–793.
15. Hodis HN, Kramsch DM, Avogaro P, Bittolo-Bon G, Cazzolato G, Hwang J, Peterson H, Sevanian A. Biochemical and cytotoxic characteristics of an in vivo circulating oxidized low density lipoprotein (LDL-). J Lipid Res. 1994;35:669–677.[Abstract]
16. Kotani K, Maekawa M, Kanno T, Kondo A, Toda N, Manabe M. Distribution of immunoreactive malondialdehyde-modified low-density lipoprotein in human serum. Biochim Biophys Acta. 1994;1215:121–125.[Medline] [Order article via Infotrieve]
17. Wang H, Chen S, Kong X, Wang X, Chang G, Xu S, Luo Z, Xie Y. Quantitation of plasma oxidatively modified low density lipoprotein by sandwich enzyme linked immunosorbent assay. Clin Chim Acta. 1993;218:97–103.[Medline] [Order article via Infotrieve]
18.
Juul K, Nielsen LB, Munkholm K, Stender S, Nordestgaard
BG. Oxidation of plasma low-density lipoprotein accelerates its
accumulation and degradation in the arterial wall in vivo.
Circulation. 1996;94:1698–1704.
19. Wagner JD, Clarkson TB, St Clair RW, Schwenke DC, Shively CA, Adams MR. Estrogen and progesterone replacement therapy reduces low density lipoprotein accumulation in the coronary arteries of surgically postmenopausal cynomolgus monkeys. J Clin Invest. 1991;88:1995–2002.
20.
Wagner JD, Zhang L, Williams JK, Register TC, Ackerman
DM, Witta B, Clarkson TB, Adams MR. Esterified estrogens with and
without methyltestosterone decrease arterial LDL
metabolism in cynomolgus monkeys. Arterioscler Thromb
Vasc Biol. 1996;16:1473–1480.
21. Maziere C, Auclair M, Ronveaux MF, Salmon S, Santus R, Maziere JC. Estrogens inhibit copper and cell-mediated modifications of low density lipoprotein. Atherosclerosis. 1991;89:175–182.[Medline] [Order article via Infotrieve]
22. Negre-Salvayre A, Pieraggi MT, Mabile L, Salvayre R. Protective effect of 17 ß-estradiol against the cytotoxicity of minimally oxidized LDL to cultured bovine aortic endothelial cells. Atherosclerosis. 1993;99:207–217.[Medline] [Order article via Infotrieve]
23. Banka CL. Non-genomic actions of estrogens: estrogens as antioxidants. In: Rubanyi GM, Kauffman R, eds. Estrogen and the Vessel Wall. London, UK: Harwood Academic Publications; 1997.
24. Sack MN, Rader DJ, Cannon RO 3rd. Oestrogen and inhibition of oxidation of low-density lipoproteins in postmenopausal women. Lancet. 1994;343:269–270.[Medline] [Order article via Infotrieve]
25.
Keaney JF Jr, Shwaery GT, Xu A, Nicolosi RJ, Loscalzo
J, Foxall TL, Vita JA. 17ß-Estradiol preserves
endothelial vasodilator function and limits low-density
lipoprotein oxidation in hypercholesterolemic swine.
Circulation. 1994;89:2251–2259.
26. Olson ME, Bruce J. Ovariectomy, ovariohysterectomy and orchidectomy in rodents and rabbits. Can Vet J. 1986;27:523–527.[Medline] [Order article via Infotrieve]
27.
Brand K, Banka CL, Mackman N, Terkeltaub RA, Fan S,
Curtiss LK. Oxidized LDL enhances lipopolysaccharide-induced
tissue factor expression in human adherent monocytes.
Arterioscler Thromb. 1994;14:790–797.
28. Berliner JA, Territo MC, Sevanian A, Ramin S, Kim JA, Bamshad B, Esterson M, Fogelman AM. Minimally modified low density lipoprotein stimulates monocyte endothelial interactions. J Clin Invest. 1990;85:1260–1266.
29.
Pitas RE, Innerarity TL, Weinstein JN, Mahley RW.
Acetoacetylated lipoproteins used to distinguish fibroblasts
from macrophages in vitro by fluorescence microscopy.
Arteriosclerosis. 1981;1:177–185.
30.
Roberts KA, Rezai AA, Pinkerton KE, Rutledge JC. Effect
of environmental tobacco smoke on LDL accumulation in the artery wall.
Circulation. 1996;94:2248–2253.
31. Rutledge JC, Pagakis SN. Extravascular distribution of low density lipoprotein and dextran in a high permeability state. Microvasc Res. 1990;40:137–156.[Medline] [Order article via Infotrieve]
32.
Roberts KA, Woo MM, Rutledge JC. Nitric oxide mediates
LDL uptake in the artery wall in response to high concentrations of
17ß-estradiol. Arterioscler Thromb Vasc Biol. 1997;17:2123–2131.
33. Shille VM, Lundstrom KE, Stabenfeldt GH. Follicular function in the domestic cat as determined by estradiol-17ß concentrations in plasma: relation to estrous behavior and cornification of exfoliated vaginal epithelium. Biol Reprod. 1979;21:953–963.[Abstract]
34.
Gilligan DM, Badar DM, Panza JA, Quyyumi AA, Cannon RO
3rd. Acute vascular effects of estrogen in postmenopausal women.
Circulation. 1994;90:786–791.
35. Wagner JD, Cefalu WT, Anthony MS, Litwak KN, Zhang L, Clarkson TB. Dietary soy protein and estrogen replacement therapy improve cardiovascular risk factors and decrease aortic cholesteryl ester content in ovariectomized cynomolgus monkeys. Metabolism. 1997;46:698–705.[Medline] [Order article via Infotrieve]
36.
Barrett-Connor E. Sex differences in coronary
heart disease: why are women so superior? the 1995 Ancel Keys Lecture.
Circulation. 1997;95:252–264.
37. Berceli SA, Warty VS, Sheppeck RA, Mandarino WA, Tanksale SK, Borovetz HS. Hemodynamics and low density lipoprotein metabolism: rates of low density lipoprotein incorporation and degradation along medial and lateral walls of the rabbit aorto-iliac bifurcation. Arteriosclerosis. 1990;10:686–694.[Abstract]
38.
Haarbo J, Nielsen LB, Stender S, Christiansen C. Aortic
permeability to LDL during estrogen therapy: a study in
normocholesterolemic rabbits. Arterioscler
Thromb. 1994;14:243–247.
39.
Hoffman RM, Garewal HS. Antioxidants and the prevention
of coronary heart disease. Arch Intern Med. 1995;155:241–246.
40.
Walzem RL, Watkins S, Frankel EN, Hansen RJ, German JB.
Older plasma lipoproteins are more susceptible to oxidation: a linking
mechanism for the lipid and oxidation theories of atherosclerotic
cardiovascular disease. Proc Natl Acad Sci
U S A. 1995;92:7460–7464.
41.
Pang L, Sivaram P, Goldberg IJ. Cell-surface
expression of an amino-terminal fragment of apolipoprotein B increases
lipoprotein lipase binding to cells. J Biol Chem. 1996;271:19518–19523.
42. Huber LA, Scheffler E, Poll T, Ziegler R, Dresel HA. 17ß-Estradiol inhibits LDL oxidation and cholesteryl ester formation in cultured macrophages. Free Radic Res. 1990;8:167–173.
43. Walsh BA, Rutledge JC. Effects of female sex hormones on LDL accumulation in the artery wall. FASEB J. 1997;11:A151. Abstract.
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