© 1997 American Heart Association, Inc.
Articles |
Nitric Oxide Mediates LDL Uptake in the Artery Wall in Response to High Concentrations of 17ß-Estradiol
From the Division of Cardiovascular Medicine and the Department of Nutrition, University of California, Davis.
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Abstract |
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Abstract Female sex hormones are known to affect lipoprotein flux in the artery wall and atherosclerosis. However, the mechanisms of these artery wall effects are unclear. To examine the effect of 17ß-estradiol (estradiol) on LDL uptake in the artery wall, we developed an isolated perfused rat carotid artery model from ovariectomized rats. LDL flux in the artery wall was measured by quantitative fluorescence microscopy before and after treatment with estradiol (0.001 to 10 000 nmol/L). Dose-response experiments showed no significant difference in the rate of LDL uptake when arteries were perfused with estradiol at physiological concentrations (0.001 to 1 nmol/L) compared with control perfusions. However, higher concentrations of estradiol (10 to 10 000 nmol/L) significantly increased the rate of LDL uptake in isolated arteries. Artery lumen volume significantly increased with perfusion of estradiol (1 to 100 nmol/L) but decreased after perfusions of higher concentrations of estradiol (1000 to 10 000 nmol/L). Additional studies were performed to examine mechanisms of estradiol-mediated increases in LDL uptake. The effect of estradiol (10 nmol/L) on the rate of LDL uptake was blocked by nitric oxide synthase inhibitors. However, the estrogen receptor antagonist tamoxifen did not block the effects of estradiol on the rate of LDL uptake. Our study indicates that modulation of LDL uptake in the artery wall by estradiol is concentration dependent. High concentrations of estradiol increase LDL uptake by production of endothelium-derived nitric oxide. These observations suggest that increased nitric oxide production compromises endothelial layer barrier function to increase LDL uptake in the artery wall.
Key Words: estrogen • artery • nitric oxide • nitric oxide synthase • low-density lipoprotein
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Introduction |
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Recent studies have indicated that female sex hormone replacement therapy protects women from developing atherosclerosis in medium and large arteries.1 2 3 A number of investigators have shown that estrogens have direct physiological effects on cardiovascular regulation. For example, some estrogens modulate vasomotor tone by endothelium-dependent or -independent mechanisms.4 5 6 7 8 9 10 11 Also, recent data indicate that estrogens modulate LDL flux and metabolism in the artery wall.12 13 14 However, mechanisms related to estrogen effects on LDL uptake in the artery wall are incompletely understood.
Estrogens have complex effects on macromolecule flux in the vascular wall. Previous investigations showed that estrogens in high concentrations increased macromolecule permeability (for example, plasma proteins) in a number of tissues, including uterus and brain.15 16 17 18 Other studies indicate that estrogen replacement therapy affects LDL flux in the artery wall. Estrogen and progesterone replacement therapy suppressed accumulation of 125I-tyramine cellobiose–LDL in coronary arteries of postmenopausal monkeys fed a high-cholesterol diet.12 Arterial content of 125I-tyramine cellobiose–LDL reflects undegraded LDL and products of LDL degradation. Furthermore, Haarbo et al13 observed significant reduction in aortic accumulation of cholesterol in ovariectomized, cholesterol-fed rabbits that were given estradiol alone or estradiol plus norethisterone acetate or levonorgesterel. The same group measured aortic permeability to 125I-LDL in normocholesterolemic, nonatherosclerotic rabbits. Aortas were removed 3 hours after infusion, and the aortic permeability to LDL was calculated from the radioactivity in the plasma and aortic intima/inner media. They found no significant differences between the groups receiving estradiol or placebo.14 Interestingly, preliminary data indicate that permeability to LDL was reduced at susceptible sites in male rabbit abdominal aortas compared with female.19 The divergence of the results of these studies may be related to plasma or tissue estrogen concentration effects, the anatomic site in the artery wall (susceptible or nonsusceptible site for atherosclerosis), and/or the presence or absence of hyperlipidemia or atherosclerosis. Further delineation of the mechanisms by which estrogens modulate arterial LDL flux is needed.
To examine mechanisms of LDL flux in the artery wall, we used an isolated rat carotid artery model from ovariectomized animals. These studies enabled us to measure LDL uptake during perfusion of estradiol over a range of concentrations from physiological to supraphysiological and to examine mechanisms of estradiol effects on LDL flux in the artery wall. Our findings indicate estradiol has direct effects on LDL uptake that are rapid, concentration-dependent, mediated by nitric oxide production, and can result in increased endothelial layer permeability.
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Methods |
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Fluorescent Solutes
LDL was isolated from human male donors by preparative ultracentrifugation20 and labeled with the fluorescent hydrocarbon probe DiI as previously described.21 DiI exchanges with lipoprotein phospholipid and does not transfer the fluorescent probe, because the two octadecyl moieties make the compound hydrophobic. LDL labeled with this method does not alter its binding capacity.22 The spectral properties of DiI are the following: excitation maximum, 540 nm and emission maximum, 556 nm. Dichroic filter sets (G-2A) were used to capture the fluorescence emitted from DiI.
To compare DiI-LDL flux with other water-soluble, nonlipid reference molecules, we used two fluorescently labeled dextrans. To examine a large molecule that is similar in size to LDL, dextran (2 000 000 MW) labeled with FITC was obtained from Sigma Chemical Company. FITC has an excitation maximum at 489 nm and maximal emission at 515 nm. B-2A filter sets captured FITC fluorescence. A smaller dextran (76 000 MW) labeled with TRITC (Sigma) was used to increase the sensitivity of changes in endothelial layer permeability. TRITC has a maximal excitation at 554 nm and maximal emission at 573 nm. G-2A filters sets were used to capture the emitted fluorescence.
Chemicals and Materials
The composition of Krebs-Henseleit solution in
mmol/L was: NaCl 116, KCl 5, CaCl2/H2O
2.4, MgCl2 1.2, NH2PO4 1.2, and
glucose 11. 17ß-Estradiol (1,3,5[10]-estratriene-3,17ß-diol; lot
No. 64H1264), L-NMMA (lot No. 84H4037), L-NAME (lot No. 14H0135),
D-NAME (lot No. 113H0909), testosterone (4-androsten-17ß-ol-3-one;
lot No. 11H4061), and tamoxifen
(trans-1-(4-ß-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1-ene;
lot No. 93H0786) were obtained from Sigma.
Animal Care
Female Sprague-Dawley rats (52 animals; 100 to 150 g)
obtained from Charles River Breeding Laboratories, Wilmington, Mass,
were used in these experiments. Animals were individually housed in
plastic cages with stainless steel covers. They were given tap water
and Purina laboratory rodent chow ad libitum. The animal housing
facility and all procedures were approved by the Animal Use Committee
at the University of California, Davis.
Ovariectomy
Rats were anesthetized with an IP injection of
ketamine (80 mg/kg) and xylazine (10 mg/kg). All
surgeries were performed in a sterilized section of the laboratory, and
instruments were autoclaved before surgery. A 0.5- to 1.0-cm
longitudinal incision was made in the midline area of the back from the
second to fifth lumbar vertebrae with a scalpel blade.23 A
small peritoneal incision was made, and the ovaries were located. Once
the ovaries were isolated, they were tied off at the tip of the uterus
and surgically removed. Vexaband tissue glue and sutures (absorbable
3-0, Dexon II) were used to close incisions. Then, animals were given
an IM injection of an antibiotic (enrofloxacin; 10 mg/kg) and
placed on a heating pad until recovery.
Experimental Protocol
Four weeks after ovariectomy, the arterial perfusion
experiments were performed. Rats were anesthetized with
injection of sodium pentobarbital (35 mg/kg, IP). After the rat
was anesthetized, an anterior 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 before 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 in a perfusion chamber.
Throughout the surgery, the artery was superfused with Krebs-Henseleit
solution. All Krebs-Henseleit solutions were gassed with 95%
O2 and 5% CO2. The artery was perfused with
Krebs-Henseleit solution plus 0.1% BSA. To reduce the number of
animals needed for these experiments, both carotid arteries were
isolated and used for perfusion.
After surgery, blood was collected from each animal via the right atrium using a 22-gauge needle and a heparinized syringe. Blood was transferred to vacuum 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 (UC Davis) for analysis of estradiol concentration by radioimmunoassay. The mean estradiol level 4 weeks postovariectomy was 11.2±1.4 pg/mL. Physiological plasma concentrations of estradiol in rats during normal estrous cycles are 30 to 500 pg/mL.24
Carotid Artery Perfusion Protocol
The perfused artery was situated in a superfusate
chamber on the microscope stage as we have described
previously.25 The Krebs-Henseleit solution was conducted
from a reservoir via tubing through a heating coil. From the heating
coil, the superfusate solution traveled via tubing to the
superfusate chamber. The superfusate solution in the
chamber was maintained at 37°C throughout the experiment.
The perfusate solution consisting of Krebs-Henseleit solution plus 0.1% BSA entered the artery through the proximal cannula and exited the artery through the distal cannula. By using three-way stopcocks and two parallel sets of tubing, the artery was perfused with either the nonfluorescent solution (Krebs-Henseleit solution plus 0.1% BSA) or the fluorescent solution (Krebs-Henseleit solution plus 0.1% BSA plus fluorescently labeled molecules [DiI-LDL]). The arteries were perfused antegrade by a Manostat peristaltic pump (Harvard). To maintain identical perfusion pressures and to convert peristaltic flow to laminar flow, syringes partially filled with fluorescent or nonfluorescent solutions (Windkessel chambers) were interposed between the pump and artery. The syringes were 50% filled with fluorescent or nonfluorescent solutions and maintained at equal heights throughout the experiment. The normal flow rate in the rat carotid artery (7 mL/min) was maintained throughout the course of the experiment. Periodic measurements of pH and partial pressures of O2 and CO2 were obtained during the course of the experiment to maintain physiological conditions.
Experimental Rig
Fluorescence measurements and imaging of the arteries
were performed simultaneously, using two experimental rigs
positioned side by side. One rig for measuring LDL flux consisted of a
Nikon MII upright microscope with a dual optical path tube (Fig 1
). A Plan x4 Nikon objective (NA 0.1)
was mounted on the microscope head. Mounted vertically on the dual
optical path tube was a Nikon P1 photometer. Mounted horizontally on
the dual optical path tube was a Hamamatsu CCD camera. The
fluorescence image was transmitted by the dual optical path
tube to the photometer and a low-light television camera. The
photometer was used to measure changes in fluorescence
intensity and was connected to a chart recorder and personal
computer. Output from the camera was input into the videocassette
recorder and to a high-resolution monitor. The other rig used to
measure LDL uptake consisted of an inverted Nikon microscope
(Diaphot-TMD), 6x objective, beam splitter, Dage low-light television
camera, and Nikon P101 photometer and controller. Using this rig, the
artery was imaged from below the Plexiglas chamber.
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60% of the screen.
Thus, simultaneous measurement of fluorescence
intensity and fluorescence imaging of the artery are
possible.
Measurement of LDL Uptake and Lumen Volume
The image of each artery was projected to the photometric
measuring window (1.2 mm2). Appropriate filters were
placed in the light (mercury) pathway to epiilluminate the artery
segment perfused with fluorescently labeled molecules.
Measurements were made during and after perfusion of each carotid
artery with the solution containing the DiI-LDL. Initially, a baseline
measurement of fluorescence intensity (If) was
established while perfusing the artery with the nonfluorescent
solution (Fig 2A). Then the artery was
perfused with the DiI-LDL solution, and a step increase in
fluorescence intensity (If0) was observed. If the
perfusate concentration of the DiI-LDL solution and
perfusate flow remain constant, If0 estimates the
lumen volume.25 As the artery continued to be perfused
with the DiI-LDL solution, some of the DiI-LDL bound to the
endothelium and/or crossed into the artery wall. After
washout with the nonfluorescent solution, DiI-LDL that remained
on the luminal surface or in the artery wall were termed If
uptake. Therefore, DiI-LDL uptake in the artery wall was determined
photometrically (in millivolts) and was a measure of the number of
DiI-LDL molecules that remain on the lumen surface and in the artery
wall. The rate of LDL uptake was If uptake divided by
perfusion time.
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In our experiments, each artery was alternately perfused with the
nonfluorescent solution for 10 minutes and the DiI-LDL solution
for 10 minutes to determine control measurements of the rate of LDL
uptake. Then, experimental treatments (for example, estradiol and
testosterone) were added to both nonfluorescent and
fluorescent solutions. The rate of LDL uptake was determined
again after treatment. In each artery, two to four measurements of the
rate of LDL uptake were made during control perfusions and three to six
measurements of the rate of LDL uptake were made after a given
treatment. Thus, each artery served as its own control. (see
Table for specific protocols.)
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Statistical Analysis
In each experiment (artery), measurements of the rate of LDL
uptake were made before and after estradiol or other compounds were
added to the perfusate solutions. Therefore, each artery served
as its own control. To give each artery and each treatment equal
weight, the mean of all measurements of the rate of LDL uptake during
control perfusion and treatment were determined. All data are
presented as the mean±SEM. Paired t tests were used
to compare control and treatment measurements. When the artery received
more than one treatment, the data were analyzed by
repeated-measures ANOVA (Sigma Stat Statistical Hardware, Jandel Corp).
Significant effects were analyzed with Student-Newman-Keuls
multiple comparison test. A probability level of P<.05 was
considered statistically significant.
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Results |
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Effects of Estradiol on the Rate of LDL Uptake in the Artery Wall
After measurements of the rate of LDL uptake were made during control perfusions of DiI-LDL in rat carotid arteries (n=10), estradiol was sequentially added to both the fluorescent and nonfluorescent solutions at doses of 0.1, 1.0, 10, 100, 1000, and 10 000 nmol/L, and repeat measurements of the rate of LDL uptake were made at each concentration (Fig 2B
). ANOVA revealed a
significant main effect of estradiol on the rate of LDL uptake
(F9,63=4.43, P<.001; Table, protocol 1, and Fig 3). Post hoc tests indicated that there
was no significant difference in the rate of LDL uptake during control
perfusion and after estradiol at doses of 0.1 and 1.0 nmol/L
(mean±SEM=0.20±0.17, 0.5±0.2, and 0.72±0.3 mV/min, respectively;
P>.05). However, the higher estradiol concentrations of 10,
100, 1000, and 10 000 nmol/L significantly increased the rate
of LDL uptake (0.98±0.5, 1.29±0.49, 2.15±0.75, and 1.77±0.51
mV/min, respectively; P<.05).
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Lumen volume also was determined during control LDL perfusions and at
each of the estradiol concentrations noted above. Estradiol at
concentrations of 1.0, 10, and 100 nmol/L significantly
increased lumen volume compared with control (35±4, 35±7, 40±1, and
28±5 mV, respectively; P<.05; Fig 4). However, the 1000 and 10 000
nmol/L concentrations significantly decreased lumen volume
(21±2 and 18±2 mV, respectively; P<.05). Because changes
in lumen volume result in changes in surface area available for
exchange of LDL across the endothelial layer, we
normalized the rate of LDL uptake in each artery by dividing the rate
of LDL uptake by the calculated surface area. Normalizing for surface
area resulted in a greater increase in the rate of LDL uptake at doses
of 1000 and 10 000 nmol/L compared with control.
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To examine the possibility of reduction of the rate of LDL uptake at
lower estradiol concentrations, we compared control measurements of the
rate of LDL uptake with the rate of LDL uptake after treatment with
estradiol (0.001 nmol/L) in a separate group of arteries (n=8;
Table, protocol 2). During these perfusions, there was no change in the
rate of LDL uptake (0.15±0.07 mV/min) compared with control
(0.14±0.05 mV/min; P=.84). Also, lumen volume was not
significantly changed after perfusion of estradiol at this
concentration (control, 38±4.6 mV versus estradiol, 37±4.1 mV,
P=.62).
In these artery perfusion experiments, estradiol was put into solution with ethanol (100%). Then the estradiol plus ethanol was diluted in the perfusate solutions to an ethanol concentration of 0.01%. To examine the effect of ethanol, the rate of LDL uptake was measured during control perfusions and after ethanol (0.01%) was added to both perfusate solutions. There was no significant change in the rate of LDL uptake after ethanol was added to the perfusate compared with control perfusions (0.2±0.1 and 0.21±0.08 mV/min, respectively; P=1.00). Likewise, lumen volume was not altered after ethanol treatment (22.2±2.8 mV) compared with control perfusions (24±4.80 mV; P=.6).
Effects of Estradiol on Arterial Uptake of
Reference Molecules
To compare arterial uptake of reference nonlipid,
water-soluble molecules with LDL, we measured the rate of uptake of
fluorescently labeled neutral dextrans in perfused arteries.
Because dextran is not expected to bind to the artery wall, uptake of
dextran in the artery wall during these short-term perfusions is a
measure of endothelial layer permeability. Dextran
(2 000 000 MW) labeled with FITC was perfused into arteries (n=5;
Table, protocol 3) before and after estradiol (10 nmol/L) was
added. The rate of dextran uptake increased after addition of estradiol
to the perfusate (0.45±0.05 mV/min) compared with control
(0.15±0.01 mV/min; P<.05). A similar increase in the rate
of dextran uptake was seen with perfusion of a smaller
fluorescently labeled dextran (76 000 MW labeled with TRITC;
n=6 arteries; Table, protocol 4). The rate of dextran uptake increased
after the addition of estradiol (0.5±0.2 mV/min) compared with control
perfusions (0.2±0.01 mV/min; P<.05). These studies
indicate changes in arterial layer
endothelial permeability as a result of treatment with
high concentrations of estradiol.
Inhibition of Nitric Oxide Synthase
We tested the effect of a nitric oxide synthase
inhibitor, L-NMMA (10-6
mol/L; n=6 arteries; Table, protocol 5), on the rate of LDL
uptake in the artery wall. The control rate of LDL uptake was 0.2±0.08
mV/min. Addition of L-NMMA to the perfusate (both
nonfluorescent and fluorescent solutions) did not
significantly change the rate of LDL uptake (0.26±0.08 mV/min). Then,
estradiol (10 nmol/L) was added to the perfusate in
addition to the L-NMMA. No significant increase in the rate of LDL
uptake was observed (0.27±0.06 mV/min; P=.52).
The effect of nitric oxide synthase on LDL uptake in the artery wall
was further investigated with another inhibitor of nitric
oxide synthase, L-NAME. Using the same protocol described above, L-NAME
(10-6 mol/L) was added to the
perfusate. The rate of LDL uptake at control perfusion
(0.2±0.01 mV/min) did not significantly change after L-NAME (0.50±0.1
mV/min) or L-NAME plus estradiol (0.2±0.01 mV/min; n=8 arteries;
P=.92; Table, protocol 6).
In another group of arteries, the enantiomer of L-NAME, D-NAME
(10-6 mol/L; Table, protocol 7), was
tested. The control rate of LDL uptake was 0.14±0.09 mV/min. Addition
of D-NAME to the perfusate did not significantly increase the
rate of LDL uptake (0.33±0.14 mV/min). Addition of estradiol (10
nmol/L) to the perfusate increased the rate of LDL
uptake (0.44±0.1 mV/min). Although a trend toward increased LDL uptake
was observed after estradiol was added to the perfusate, it did
not reach statistical significance (P=.06).
To determine whether competitive inhibition of nitric oxide synthase
could be overridden by L-arginine, the substrate for nitric
oxide synthase, we performed experiments (Table, protocol 8) in which
the rate of dextran uptake was determined (0.09±0.02 mV/min) during
control perfusions of dextran labeled with TRITC (76 000 MW; 75
mg/mL). Then, L-NAME (10-6
mol/L), L-arginine (10-4
mol/L), and estradiol (10 nmol/L) were added in sequence,
and the rate of dextran uptake was again determined (0.13±0.01 mV/min;
P=.02). Thus, a surplus of L-arginine can
override the inhibition induced by L-NAME.
We compared the effect of 17
-estradiol, a stereoisomer of
17ß-estradiol, on LDL uptake in the artery wall (Table, protocol 9).
Some,18 26 but not all,27 previous studies
indicate 17-estradiol does not stimulate production of
nitric oxide, as does 17ß-estradiol. In our experiments, the rate of
LDL uptake during control perfusions (0.24±0.07 mV/min; n=4 arteries)
and after treatment with 17-estradiol (10 nmol/L; 0.26±0.1
mV/min) was not significantly different. Thus, 17ß-estradiol, but not
17-estradiol, modulated LDL uptake in the artery wall.
To further examine the effect of nitric oxide donors on LDL uptake in
the artery wall, we measured the rate of LDL uptake in the artery wall
during control perfusions (0.16±0.03 mV/min; Table, protocol 9). Then
the nitric oxide donor sodium nitrite was perfused into the artery
(Table, protocol 10), first at a concentration of
10-6 mol/L (0.2±0.04 mV/min), and the
rate of LDL uptake in the artery wall was again measured. This step was
followed by perfusion with sodium nitrite at
10-4 mol/L (0.25±0.06 mV/min). Sodium
nitrite (10-4 mol/L) significantly
increased LDL uptake compared with control (P<.05). In
addition, Fig 5 shows an artery perfused
at control conditions and after perfusion with sodium nitrite
(10-6 mol/L), using imaging techniques
that we have previously described.28 Greater LDL uptake is
noted when the artery is treated with sodium nitrite. These experiments
indicate that a nitric oxide donor in high concentration can increase
the rate of LDL uptake.
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To examine more directly the interaction of nitric oxide and
endothelial layer permeability, we performed
experiments in which nitric oxide was generated by a nitric oxide
saturation process.29 Nitric oxide was added to the
perfusate (Table, protocol 11) at 0.23 and 23 nmol/L
concentrations (Fig 6). The rates of LDL
uptake during control perfusions and after perfusion of 0.23 and 23
nmol/L were 0.24±0.14, 0.29±018, and 1.4±0.27 mV/min,
respectively. The rate of LDL uptake was significantly greater than
control during perfusion of nitric oxide (23 nmol/L;
P<.05).
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Effect of an Estrogen Receptor Antagonist
We examined the effect of tamoxifen on LDL uptake in the artery
wall. Initially, we perfused a concentration of tamoxifen
(10-6 mol/L; Table, protocol 12) that
had been tested as an estrogen receptor antagonist in a
variety of cell types.26 30 31 Tamoxifen increased the
rate of LDL uptake compared with control LDL perfusions (0.68±0.025
and 0.24±0.010 mV/min, respectively; n=10 arteries;
P<.01).
The effect of tamoxifen was determined at a lower concentration
(10-8 mol/L; Fig 5; Table, protocol 13)
in five arteries. In these arteries, the rate of LDL uptake during
control LDL perfusions and treatment with tamoxifen and tamoxifen plus
estradiol (10 nmol/L) was 0.1±0.03, 0.1±0.04, and 0.24±0.07
mV/min, respectively. The rate of LDL uptake for the tamoxifen plus
estradiol treatment was significantly greater than control or tamoxifen
treatment alone (P<.05). Thus, high concentrations of
tamoxifen had agonistic effects. At lower concentrations, in which
tamoxifen showed no agonistic effects, tamoxifen did not block the
expected increase in the rate of LDL uptake when the artery was treated
with estradiol.
Effects of Testosterone on LDL Uptake
For comparison, we examined the effect the male sex hormone
testosterone had on LDL uptake in the artery wall (Table, protocol 14).
Testosterone was added to the perfusate solutions and the rate
of LDL uptake measured in ovariectomized rats. There was a main effect
after treatment with testosterone on the rate of LDL uptake
(F9,36=7.15; P<.001). A post hoc test indicated
that testosterone at doses of 1 and 10 µmol/L did not
increase the rate of LDL uptake (0.3±0.08 and 0.4±0.04 mV/min,
respectively) compared with control (0.2±0.05 mV/min;
P>.05). However, testosterone at 100 µmol/L
significantly increased the rate of LDL uptake (0.6±0.02 mV/min;
P<.05) compared with control. Thus, only at very high
concentrations did testosterone have any effect on the rate of LDL
uptake.
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Discussion |
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Our studies indicate that physiological concentrations of estradiol did not alter artery wall LDL uptake but did induce vasorelaxation. However, supraphysiological concentrations of estradiol increased LDL uptake in normal artery walls. This investigation indicates that when the artery wall is exposed to high estradiol concentrations, the mechanism of increased LDL uptake is mediated by production of nitric oxide via nitric oxide synthase. Our studies suggest that excess production of nitric oxide may adversely affect vascular wall function.
Studies performed in a number of vascular beds demonstrate increased blood vessel permeability when vessels are perfused with high concentrations of a variety of estrogens.15 16 17 18 Also, disease states associated with high estradiol levels and increased production of nitric oxide have been characterized by altered vascular permeability. For example, increased nitric oxide production may be an important mediator in preeclamptic pregnancies.32 33 Furthermore, the ovarian hyperstimulation syndrome has been described in women undergoing assisted reproduction.34 35 Some women who undergo assisted reproduction are treated with high concentrations of gonadotropins, such as human chorionic gonadotropin. These treatments stimulate the ovaries to produce more estrogens. The ovarian hyperstimulation syndrome is characterized in part by increased vascular permeability, causing ascites and pulmonary edema. Although our data were initially surprising to us, review of the literature documents estrogen-induced increases in vascular wall permeability.
Some of the early studies of hormone replacement therapy suggested no cardiovascular benefit or increased mortality.36 37 38 39 40 In most of these studies, high oral doses of hormone replacement therapy were administered compared with current recommended doses. Also, the Coronary Drug Project showed increased risk of myocardial infarction when male patients were given high oral doses of estradiol (2.5 and 5 mg/d).41 42 Consistent with these observations, our study showed increased LDL uptake when arteries were perfused with supraphysiological concentrations of estradiol. Increased uptake of LDL in the artery wall is one of the earliest manifestations of the developing atherosclerotic plaque. Thus, high plasma concentrations of female sex hormones could potentially accelerate atherosclerosis and increase its complications.
LDL flux in the artery wall involves complex and concurrent processes. Some of these processes include endothelial layer permselectivity, diffusion or convection of LDL in the intima, binding of LDL to components of the artery wall such as glycosaminoglycans, incorporation and degradation of LDL in arterial wall cells, and efflux pathways by which LDL leaves the artery. Perturbation of any of these processes could result in increased LDL content in the artery wall. In contrast to the studies of Wagner et al12 and Haarbo et al,13 our short-term perfusion studies do not address issues of incorporation and degradation of LDL in cells. As discussed below, a nonselective change in endothelial layer permeability could account for both increased LDL and dextran uptake when the vessel was treated with high concentrations of estradiol.
Our data indicate estradiol in supraphysiological concentrations increases LDL uptake by formation of nitric oxide. A number of previous studies showed that nitric oxide can compromise endothelial layer barrier function.43 44 45 46 47 48 49 Although the mechanism accounting for increased permeability when high concentrations of estradiol are perfused is not known with certainty, the following intracellular signaling scheme is postulated to explain our results: Estradiol rapidly increases [Ca2+]i, either by increased transmembrane Ca2+ flux or release of intracellular stores.50 Increased [Ca2+]i induces constitutive nitric oxide synthase to increase nitric oxide production. Nitric oxide activates guanylate cyclase and leads to the formation of cGMP, which acts on the endothelial cytoskeleton and contractile elements. Activation of contractile elements effaces the interendothelial cell junction, effectively increasing extracellular pore size.45 This effect nonspecifically increases transendothelial macromolecular permeability through convective, water-filled pathways for molecules of diverse sizes, such as LDL and dextrans.51
The theoretical mechanisms described above presume that administration of estradiol has an almost immediate effect on nitric oxide production. Relatively few studies have investigated this possibility. Reis et al 8 showed that short-term infusion of ethinyl estradiol increased coronary flow, decreased resistance, and increased epicardial coronary artery cross-sectional area 15 minutes after infusion of ethinyl estradiol compared with placebo in postmenopausal women.8 This study is consistent with ours in that estradiol appears to have an immediate effect on the vascular wall.
Most previous studies showed endothelium-derived nitric oxide to be atheroprotective; however, some data indicate that overproduction of nitric oxide can be deleterious. For example, nitric oxide was shown to be a factor in neuronal apoptosis.52 Also, because nitric oxide is a free radical, it potentially could modify LDL, as was shown by Chang et al.53 Modified LDL will bind more avidly to other lipoproteins, glycosaminoglycans, macrophages, and smooth muscle cells; all effects that accelerate the atherosclerotic process. In addition, some reports suggest that atherosclerotic arteries have increased capacity for generation of nitric oxide by inducible nitric oxide synthase.54 The potentially damaging effect of excess nitric oxide production should be considered in the development of therapies to treat or prevent atherosclerosis.
Our studies are not inconsistent with previous studies on the effect of estradiol on the artery wall. The animal model that we used did not have high lipids and was not atherosclerotic. Our animal model is closest to the normocholesterolemic model as described by Haarbo et al.14 Both models show no significant change in permeability or uptake when physiological concentrations of estradiol are administered. In contrast, our acute studies showed increased LDL uptake when high concentrations of estradiol were administered. High concentrations of estradiol were not administered by Haarbo et al. However, studies performed in hyperlipidemic atherosclerotic models show reduced LDL accumulation when the animal is treated with estradiol and progesterone replacement.39 The mechanisms of reduction in LDL accumulation are currently being intensely investigated in a number of laboratories.
Testosterone also appears to have dose-related effects on the artery wall. Previous studies in men indicated that low testosterone levels are a risk factor for atherosclerosis,55 whereas high androgen levels promote atherosclerosis in female cynomolgus monkeys.56 Although the literature is conflicting,57 most recent data indicate that testosterone enhances arterial relaxation.56 58 Consistent with previous data,59 our study showed increased LDL uptake in the artery wall only in supraphysiological concentrations. In general, these studies suggest in males that androgens in physiological concentrations are important in maintaining normal vascular function and prevention from disease.
Conclusion
Although endothelium-derived nitric oxide is
beneficial to the artery in many ways, excessive production of
nitric oxide may be detrimental to the vascular wall and may
specifically increase LDL uptake in the artery wall. These studies
indicate that estrogen effects on the artery wall are complex,
multifaceted, and dose dependent. Appropriate clinical dosing of
estrogens is important to obtain the full
cardiovascular benefit.
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Selected Abbreviations and Acronyms |
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-nitro-D-arginine methyl ester
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Appendix 1 |
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TopAbstractIntroductionMethodsResultsDiscussionAppendix 1References |
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We compared our method for measurement of LDL uptake in the artery wall with other methods. Using our experimental preparation, LDL cholesterol uptake in the segment of perfused carotid artery can be estimated. If0 is a measure of the fluorescence intensity of the DiI-labeled perfusate in the artery lumen. The LDL cholesterol concentration of the perfusate solution is known and is assumed to remain constant in the photometric measuring window during the perfusion. The amount of LDL cholesterol in the artery lumen segment can be determined from the LDL cholesterol concentration in the lumen and the lumen volume. The volume of the artery lumen is
r2l. The radius (r) of the carotid artery can be
measured by our optical system, which is calibrated by a stage
micrometer. The length of the carotid artery in the
photometric measuring window (L) is 0.012 cm. Thus, If0
measures the fluorescence intensity of fluorescently
labeled LDL in the photometric measuring window. Our calibration
experiments show that fluorescence intensity is linearly
correlated with the volume of the artery lumen. Thus, If0
is linearly correlated with total LDL cholesterol in the
artery lumen. If uptake measures the fluorescence
intensity of DiI-LDL in the artery wall after the lumen is washed out
and is assumed to be linearly correlated to LDL cholesterol
in the artery wall. Thus, If Uptake/If0 Lumen =Artery Wall LDL Cholesterol/LDL Cholesterol
The rate of LDL uptake in the artery wall (micrograms cholesterol per gram tissue per minute) can be determined by using the known concentration of LDL cholesterol in the perfusate, the volume of the artery in the photometric window, If0, If uptake, and the weight of the artery in the measuring window. For example, a typical artery is perfused with 0.027 mg/mL LDL cholesterol. At the photometer settings used in the experiments, If0 is about 29 mV. In control LDL perfusions (10 minutes), If uptake was about 2 mV. If the measured radius of the artery is 0.05 cm and the length of the carotid artery in the measuring window is 0.12 cm, then the lumen volume is 0.0009 mL. Given the LDL cholesterol concentration of the perfusate, the total LDL cholesterol in the lumen is 0.024 µg. If If uptake is 2 mV after a 10-minute LDL perfusion and If0 is 29 mV, the LDL cholesterol uptake in the artery wall is 0.0017 µg, and the rate of uptake is 0.00017 µg/min. The weight of the 0.12-cm segment of the carotid is about 0.02 g. Thus, during control perfusions, LDL cholesterol accumulated in the artery at a rate of 0.0085 µg · g-1 · min-1 or 0.51 µg · g-1 · h-1. In a previous study, 125I-tyramine cellobiose–labeled LDL was perfused into a rabbit, and rates of LDL permeability into uniform segments of the abdominal aorta were determined.60 The rate of LDL influx per unit weight into the rabbit abdominal aorta was 0.37 µg · g-1 · h-1. This value compares favorably with the rate of LDL cholesterol uptake that we estimate in our perfused arterial experiments.
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Acknowledgments |
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This project was supported by a grant from the National Heart, Lung, and Blood Institute (K11-HL-02112). K. Roberts was supported by an NIH training grant in nutrition (No. DK07355). We thank Kristine Lewis and Allen Rezai for technical assistance. Barbara Walsh provided valuable comments about this manuscript.
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Footnotes |
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Reprint requests to John C. Rutledge, Division of Cardiovascular Medicine, TB 172, Bioletti Way, University of California, Davis, CA 95616.
Received June 12, 1996; accepted May 9, 1997.
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References |
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