© 1999 American Heart Association, Inc.
Original Contribution |
Estrogen and Tamoxifen Metabolites Protect Smooth Muscle Cell Membrane Phospholipids Against Peroxidation and Inhibit Cell Growth
From the Center for Clinical Pharmacology and Department of Medicine (R.K.D., D.G., R.A.B., E.K.J.), Department of Pharmacology (E.K.J.), Department of Environmental and Occupational Health (Y.Y.T., V.A.T., V.E.K.), and Cancer Institute (V.E.K.), University of Pittsburgh, Pittsburgh, Pa; and Clinic for Endocrinology, Department of Obstetrics and Gynecology (R.K.D.), University Hospital Zurich, Switzerland.
Correspondence to Dr Raghvendra K. Dubey, Center for Clinical Pharmacology, Department of Medicine and Pharmacology, 623 Scaife Hall, 200 Lothrop St, University of Pittsburgh Medical Center, Pittsburgh, PA 15213-2582. E-mail dubey{at}med1.dept-med.pitt.edu
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Abstract—The goal of this study was to test the hypothesis that antioxidant estrogens, by a mechanism independent of the estrogen receptor, protect phospholipids residing in the plasma membrane of vascular smooth muscle cells from peroxidation and peroxidation-induced cell growth and migration. Peroxidation of membrane phospholipids was assessed by HPLC analysis of phospholipids extracted from rat aortic vascular smooth muscle cells prelabeled with cis-parinaric acid (a fatty acid that is susceptible to peroxidation, which quenches its fluorescent properties). Incubation of cells for 2 hours with the peroxyl radical donor 2,2'-azobis-2,4-dimethylvaleronitrile (AMVN) caused peroxidation of all measured membrane phospholipids. This effect was attenuated by pretreating cells for 15 minutes with 50 to 5000 ng/mL of 2-hydroxyestradiol (strong antioxidant but weak estrogen-receptor ligand) or 4-hydroxytamoxifen (strong antioxidant and potent estrogen-receptor ligand), but not by estrone or droloxifene (both weak antioxidants but potent estrogen-receptor ligands). Moreover, pretreatment of cells for 20 hours with physiological concentrations (0.3 ng/mL) of 2-hydroxyestradiol or pharmacologically relevant concentrations of 4-hydroxytamoxifen (40 ng/mL) also decreased AMVN-induced phospholipid peroxidation. Both 2-hydroxyestradiol and 4-hydroxytamoxifen were as effective as 2,2,5,7,8-pentamethyl-6-hydrochromane (an antioxidant homolog of vitamin E) in attenuating AMVN-induced peroxidation of membrane phospholipids. Also, physiological concentrations of 2-hydroxyestradiol, but not estrone, and pharmacologically relevant concentrations of 4-hydroxytamoxifen attenuated AMVM-induced DNA synthesis, cell proliferation, and cell migration. These studies demonstrate in vascular smooth muscle cells that antioxidant estrogens via a non-estrogen receptor–dependent mechanism attenuate peroxidation of membrane phospholipids and peroxidation-induced cell growth and migration.
Key Words: estrogen • metabolism • antioxidant • cardiovascular disease • free radical
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Although it is well established that estrogens are cardioprotective in postmenopausal women,1 2 3 4 5 the mechanisms of estrogen-induced reduction in the risk of coronary artery disease remain unclear. Because a complete understanding of estrogen-mediated cardioprotection may allow the development of novel agents for the prevention and treatment of coronary artery disease in both women and men, further exploration in this regard is warranted.
One explanation that has been advanced to account in part for the cardioprotective effects of estrogens is the antioxidant hypothesis. Two main lines of evidence support this hypothesis. First, estrogens or their metabolites contain 1 or more phenolic functional groups, and the phenol moiety can scavenge free radicals.6 For example, 17ß-estradiol not only contains a phenolic functional group, but also is metabolized to 2-hydroxyestradiol, a catechol estrogen and potent antioxidant. Although tamoxifen, another cardioprotective estrogen-receptor ligand,7 does not contain a phenolic group, it can be metabolized to the antioxidant phenolic compound 4-hydroxytamoxifen.8 Second, estrogen and tamoxifen attenuate free radical–induced oxidation of plasma LDL,8 9 10 11 12 13 although diminished formation of oxidized LDL per se cannot explain all the cardiovascular benefits of estrogens, because restenosis after angioplasty and bypass surgery, a process that is independent of oxidized LDL, is attenuated by estrogens.14 15 16 17
It is conceivable that estrogens could also protect critical cellular structures from oxidative damage and that this may contribute to the cardioprotective effects of estrogens. In this regard, it is well established that free radicals oxidize membrane lipids and integral membrane proteins18 19 and that such oxidations can alter signal-transduction mechanisms controlling cell migration and proliferation,20 21 processes critical to vascular neointimal formation. Moreover, because estrogens are lipophilic, high local concentrations of these compounds would exist in the biophase of the lipid bilayer during hormone replacement therapy. Thus, the hypothesis that estrogens may attenuate oxidation of membrane components has a strong a priori rationale. Accordingly, the main objective of the present study was to test the hypothesis that antioxidant estrogens, by a mechanism independent of the estrogen receptor, protect phospholipids residing in the plasma membrane of vascular smooth muscle cells from free radical–induced peroxidation and peroxidation-induced cell growth and migration.
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Materials and Methods |
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cis-Parinaric acid (Z-9,E-11,E-13,Z-15–octadecatetraenoic acid) was purchased from Molecular Probes, Inc. The purity of each batch of cis-parinaric acid used was determined by UV spectrophotometry (Shimadzu UV 160U spectrophotometer) using a molar extinction at 304 nm in ethanol of 80x103 (mol/L)-1 · cm-1. 2,2'-Azobis (2,4-dimethylvaleronitrile) (AMVN) was procured from Polysciences, Inc. 2,2'-Azobis (2-aminodinopropane) dihydrochloride (AAPH) was purchased from Wako Chemicals USA Inc. Fatty acid-free human serum albumin (hSA), phospholipids, 5,5'-dithiobis(2-nitrobenzoic acid), estrone, glutathione, glutathione-peroxidase, cumene hydroperoxide, 2-mercaptoethylamine (cysteamine) hydrochloride, Tween 20, sodium molybdate, malachite green base, butylated hydroxytoluene, and 3-[4,5-dimethylthiozol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) were purchased from Sigma Chemical Co. Methanol, chloroform, hexane, 2-propanol, and water were purchased from Aldrich Chemical Co. Deferoxamine mesylate was purchased from Ciba. 2,2,5,7,8-Pentamethyl-6-hydrochromane (PMC) was a generous gift from Eisai Co. 4-Hydroxytamoxifen and droloxifene were obtained from Research Biochemicals International. 2-Hydroxyestradiol was purchased from Steraloids Inc. Phenol red–free DMEM and DMEM/F12 medium, HBSS, penicillin, streptomycin, 0.25% trypsin-EDTA solution, and all tissue culture instruments were purchased from Gibco Laboratories. FCS was obtained from HyClone Laboratories Inc. All other chemicals used were of tissue-culture grade or best grade available.
General Experimental Approach
To test the hypothesis that antioxidant estrogens inhibit
oxidation of membrane structures, it was necessary to measure free
radical–induced oxidation of membrane lipids in intact vascular smooth
muscle cells. In this regard, we used the novel strategy of monitoring
fluorescence intensity of membrane-incorporated
cis-parinaric acid, a naturally occurring 18-carbon fatty
acid that is highly susceptible to free radical–induced peroxidation,
which markedly quenches its fluorescence.22
Because cis-parinaric acid fluorescence is lost
permanently on oxidation of the conjugated double-bond system, repair
mechanisms, which are normally present in live cells, cannot mask
the oxidative events. This is true even if cis-parinaric
acid is oxidized and replaced with unoxidized fatty acids, since the
evidence for oxidation remains as loss of cis-parinaric acid
fluorescence. This characteristic is not found in assays
currently used in cultured cells and animal models. Furthermore, use of
cis-parinaric acid also enables the localization of the
effects of oxidative stress on different classes of phospholipids.
Peroxyl radicals were generated by the addition of
AMVN,23 and fluorescence intensity of the
reporter molecule, cis-parinaric acid, was monitored in
several membrane phospholipid components in the absence and presence of
2-hydroxyestradiol, 4-hydroxytamoxifen, estrone, and droloxifene. These
particular estrogens were selected because 2-hydroxyestradiol and
4-hydroxytamoxifen are strong antioxidants but different in their
potency as estrogen-receptor ligands
(4-hydroxytamoxifen>2-hydroxyestradiol), whereas estrone and
droloxifene are weak antioxidants but potent estrogen-receptor
ligands.6 8 11 We anticipated that using a diverse
set of estrogens with different antioxidant and estrogen-receptor
ligand affinities would allow inferences regarding whether a given
effect was dependent or independent of estrogen receptors. Moreover, we
used PMC, a potent antioxidant, as a reference benchmark for evaluating
the antioxidant potency of the estrogens tested in this study. We also
investigated the selected estrogens with regard to their ability to
attenuate peroxyl radical–induced changes in endogenous
thiols (reduced glutathione [GSH] and protein sulfhydryls) in
vascular smooth muscle cells, using in this case AAPH23 as
the oxidant stress. The addition of these experiments allowed us to
contrast the antioxidant effects of estrogens at 2 important targets
for oxidative changes. Finally, to evaluate the
physiological relevance of the antioxidant effects
of estrogens, we conducted cell proliferation,
[3H]thymidine incorporation, and cell migration
studies to determine whether peroxyl radicals induce smooth muscle cell
growth and migration and whether the selected estrogens inhibit free
radical–induced DNA synthesis, cell proliferation, and cell
migration.
Aortic Smooth Muscle Cell Culture
Male Sprague-Dawley rats (Charles River, Wilmington, MA)
weighing 150 to 200 g were fed standard rat chow and tap water ad
libitum. Aortic smooth muscle cells were cultured as explants from the
lower abdominal aortas that were obtained from
ether-anesthetized rats after a midline abdominal incision
including the diaphragm and as described previously.24
Smooth muscle cell purity was characterized by
immunofluorescence staining with specific
anti–smooth muscle 
-actin monoclonal antibodies and by
morphological criteria specific for smooth muscle as described in
detail previously.25 Smooth muscle cells between the
second and third passages were used for the cellular lipid peroxidation
and thiol (GSH) oxidation studies.
Incorporation of cis-Parinaric Acid Into Smooth
Muscle Cell Phospholipids
cis-Parinaric acid was incorporated into smooth
muscle cells by addition of cis-parinaric acid complexed
with hSA [cis-parinaric acid (500 µg, 1.8 µmol) in
25 µL of DMSO and hSA (50 mg, 760 nmol) in 1 mL of PBS (in
mmol/L, 137 NaCl, 2.7 KCl, 1.5
KH2PO4, and 8
Na2HPO4, pH 7.4)] to the
cell suspensions. Briefly, confluent monolayers of smooth muscle cell
were dislodged by trypsinization, washed once with DMEM
containing 10% FCS, and washed subsequently with DMEM alone. Before
the experiments, the smooth muscle cells were rinsed twice with L1210
buffer containing (in mmol/L) 115 NaCl, 5 KCl, 1
MgCl2, 5
NaH2PO4, 10 glucose, and 25
HEPES (pH 7.4). Cells were diluted to a density of
1.0x106 cells/mL and then incubated with
cis-parinaric acid-hSA complex (final concentration of 5
µg/mL cis-parinaric acid) in L1210 buffer at 37°C in the
dark and under air. The smooth muscle cells were incubated for
different time periods (30, 60, 120, and 180 minutes), and the time
course of cis-parinaric acid incorporation into cellular
phospholipids was evaluated after washing the cells twice with L1210
buffer with and without hSA (0.5 mg/mL). Trypan blue exclusion tests
for cell viability were conducted in parallel by taking aliquots of
smooth muscle cells treated for different time periods.
AMVN-Induced Peroxidation of Membrane Lipids
AMVN, a lipid-soluble azo- compound that is a thermally
activated initiator of peroxyl radicals, was used to induce
lipid peroxidation. Because of its hydrophobic character, AMVN
partitions into the acyl lipid region of membranes and generates
radicals26 that do not escape from the hydrophobic lipid
environment.23 Moreover, oxidation of phospholipids by
AMVN tends to be nonselective, so in principle it is possible to
determine whether particular phospholipid classes are intrinsically
more susceptible to oxidation than others. Smooth muscle cells were
incubated for 2 hours with cis-parinaric acid, and
cis-parinaric acid–labeled cells were subsequently
incubated in the presence of AMVN (500 µmol/L) in L1210 buffer
at 37°C in the dark in air for 2 hours. Appropriate amounts of
phenolic compounds (4-hydroxytamoxifen, droloxifene, estrone,
2-hydroxyestradiol, or PMC) or cysteamine were added to the incubation
medium 15 minutes before addition of AMVN. After incubation for 2
hours, aliquots of cell suspension were taken for both determination of
cell viability and lipid analysis.
To evaluate whether long-term treatment of cells with 4-hydroxytamoxifen, estrone, 2-hydroxyestradiol, and PMC prevents AMVN-induced peroxidation of smooth muscle cells, trypsinized cells were suspended in DMEM containing 0.4% albumin and treated for 20 hours with or without 0.3 to 30 ng/mL of the above agents. Following incubation the smooth muscle cells were loaded with cis-parinaric acid (as described above) and subsequently incubated with AMVN (250 µmol/L) for 3 hours. After incubation, aliquots of cell suspension were taken for both determination of cell viability and lipid analysis.
Extraction of Cell Lipids
Total lipids were extracted by using a procedure adapted from
that of Folch et al.27 Briefly, methanol (2 mL) containing
butylated hydroxytoluene (0.1 mg) was added to the cell suspension
(1x106 cells). The suspension was mixed with
chloroform (4 mL) and kept for 1 hour under a nitrogen atmosphere on
ice in the dark to ensure complete extraction. After addition of 0.1
mol/L NaCl (1 mL) and vortex mixing (still under a nitrogen
atmosphere), the chloroform layer was separated by
centrifugation (1500g, 5 minutes). The
chloroform was evaporated with a stream of nitrogen, and the lipid
extract was dissolved in 4:3:0.16 (vol/vol) 2-propanol–hexane–water
(0.2 mL). Control experiments demonstrated that the procedure recovered
>95% of cell phospholipids.
HPLC Analysis of Cell Lipids
Lipid extracts were separated with an ammonium acetate gradient
using normal-phase HPLC by slight modification of the method of Geurts
van Kessel et al.28 Briefly, a 5-µm Supelcosil LC-Si
column (4.6x250 mm) was used with the following mobile phase
flowing at 1 mL/min: solvent A (2-propanol–hexane–water 57:43:1
vol/vol), solvent B (2-propanol–hexane–40 mmol/L aqueous
ammonium acetate, pH 6.7), 0- to 3-minute linear gradient from 10%
solvent B to 37% solvent B, 3- to 15-minute isocratic elution
at 37% solvent B, 15- to 23-minute linear gradient to 100% solvent B,
and 23- to 45-minute isocratic elution at 100% solvent B. A Shimadzu
high-performance liquid chromatograph (model LC-600)
equipped with an in-line configuration of fluorescence (model
RF-551) and absorbance (model SPD-10A V) detectors was used. The
effluent was monitored at 205 nm to gauge separation of lipids.
Fluorescence emitted by cis-parinaric acid in
eluates was monitored fluorometrically at 420-nm emission and 324-nm
excitation, and the data were processed with Shimadzu EZChrom
software.
Determination of Lipid Phosphorus in Lipid Extracts
Lipid phosphorus in lipid extracts was estimated
spectrophotometrically at 660 nm as described by Chalvardjian and
Rubnicki,29 with minor modifications. Aliquots of lipid
extract were pipetted into test tubes, and the solvent was evaporated
to dryness under a stream of nitrogen. Perchloric acid (50 µL) was
then added to samples, and the mixtures were incubated for 20 minutes
at 170°C to 180°C. After the tubes were cooled, 0.4 mL of distilled
water was added to each tube followed by 2 mL of sodium
molybdate–malachite green reagent (4.2% sodium molybdate in 5.0N
hydrochloric acid/0.2% malachite green, 1:3 vol/vol). Without delay,
80 µL of 1.5% Tween 20 was added, mixed immediately to stabilize the
color, and read spectrophotometrically at 660 nm.
High-Performance Thin-Layer Chromatograph
Analysis of Cell Lipids
Different classes of phospholipid extracted from smooth muscle
cells were separated by 2-dimensional high-performance
thin-layer chromatography on silica G plates (5x5 cm,
Whatman). The plates were first developed with a solvent system
consisting of chloroform:methanol:28% ammonium hydroxide, (65:25:5,
vol/vol). After drying, the plates were developed in the second
dimension with a mixture of chloroform, acetone, methanol, glacial
acetic acid, and water (50:20:10:10:5, vol/vol). The phospholipids were
then visualized by exposing the plates to iodine vapor and the spots
identified by comparison with migration of authentic phospholipid
standards. The spots identified by iodine staining were scraped off the
plates, and the silica acid was transferred to tubes. Lipid phosphorus
in individual phospholipid spots of the HPLC plates were extracted and
quantified spectrophotometrically with the method of Arduini et
al.30 The identity of each phospholipid was established by
comparison with the Rf values measured for authentic
standards.
AAPH-Induced Oxidation of Intracellular Thiols and
Glutathione
AMVN-derived peroxyl radicals do not escape the lipid
environment of the membrane23 ; hence, AMVN is not a good
agent with which to study the effects of peroxyl radicals on
intracellular glutathione oxidation. Therefore, we used AAPH, an analog
of AMVN that is a water-soluble azo- initiator. Smooth muscle cells
were incubated with AAPH (20 mmol/L in L1210 buffer, pH 7.4) for 2
hours at 37°C in the absence or in the presence of different
concentrations of the phenolic compounds (4-hydroxytamoxifen,
droloxifene, estrone, 2-hydroxyestradiol, or PMC). The reaction was
terminated by cooling the samples to 4°C. In parallel to the above
vascular smooth muscle cell experiments, we also investigated the
direct effects of the phenolic compounds on the oxidation of pure GSH.
Briefly, glutathione (50 µmol/L) was incubated for 10 minutes in
phosphate buffer (50 mmol/L), pH 7.4, both in the absence and in
the presence of phenolic compound, and the levels of glutathione were
assayed.
Determination of Glutathione Concentration in Smooth Muscle
Cells
Glutathione concentration in smooth muscle cells was determined
by estimating the difference in 5,5'-dithiobis(2-nitrobenzoic
acid)-titratable thiols31 in the presence and in the
absence of glutathione peroxidase and cumene hydroperoxide. Smooth
muscle cells (5x106 cells) were sonicated and
then incubated with glutathione peroxidase (1.94 U/µL), cumene
hydroperoxide (333 µmol/L), and deferoxamine
mesylate (100 µmol/L) for 30 minutes at 25°C. An aliquot of
cell lysate was then added to 200 µmol/L
5,5'-dithiobis(2-nitrobenzoic acid), and the precipitated protein was
separated by centrifugation for 5 minutes,
10 000g at 4°C. The absorbance of supernatant was
determined spectrophotometrically at 412 nm.
Growth Studies
[3H]Thymidine incorporation, cell
number, and cell migration studies were conducted to investigate the
effects of agents on AMVN-induced DNA synthesis, cell proliferation,
and cell migration. Smooth muscle cells were plated at a density of
4x104 cells/well in 24-well tissue-culture
dishes and allowed to grow to subconfluence in DMEM/F12 (phenol
red–free) medium containing 10% FCS (steroid free and delipidated)
under standard tissue culture conditions. The cells were then growth
arrested by feeding with DMEM (phenol red free) containing 0.4%
albumin for 48 hours. For DNA synthesis, growth was initiated
by treating growth-arrested cells for 20 hours with DMEM containing
0.5% FCS and supplemented with AMVN (1 to 100 µmol/L) and
containing or lacking estrone, 2-hydroxyestradiol, 4-hydroxytamoxifen,
or droloxifene. After 20 hours of incubation, the treatments were
repeated with freshly prepared solutions but supplemented with
[3H]thymidine (1 µCi/mL; ICN Biomedicals) for
an additional 4 hours. The experiments were terminated by washing the
cells twice with Dulbecco's PBS and twice with ice-cold
trichloroacetic acid (10%). The precipitate was solubilized in 500
µL of 0.3N NaOH and 0.1% SDS after incubation at 50°C for 2 hours.
Aliquots from 4 wells for each treatment with 10 mL of scintillation
fluid were counted in a liquid scintillation counter. For cell number
experiments, smooth muscle cells were allowed to attach overnight, were
growth arrested for 48 hours, were treated every 24 hours for 4 days,
and on day 5 were dislodged and counted on a Coulter counter.
Cell Migration Studies
Modified Boyden chambers (Neuro Probe Inc) were used to
evaluate the effects of AMVN-derived peroxyl radicals on smooth muscle
cell migration as previously described.24 Briefly,
confluent monolayers of smooth muscle cells were growth arrested by
feeding with DMEM supplemented with 0.4% albumin for 48 hours.
Growth- arrested smooth muscle cells were trypsinized, washed, and
suspended at a concentration of 1x106 cells/mL
in fresh DMEM containing 0.4% albumin and supplemented with
AMVM (50 µmol/L) and containing or lacking 0.3 to 30 ng/mL
estrone, 2-hydroxyestradiol, 4-hydroxytamoxifen, or PMC. Following
incubation, the smooth muscle cells (50 000 cells/50 µL; 6000
cells/mm2) were layered on the top chamber, and
DMEM containing the respective treatments plus 0.5% FCS was added to
the lower chamber. After 6 hours of incubation, the membranes were
removed, the nonmigrated cells on the top surface wiped, and the
migrated cells fixed and stained in Dif Quick stain (Baxter Scientific
Corp). The migrated cells on the lower surface of the membrane were
counted manually at 6 different areas of the membrane.
Cell Viability Assays
Trypan blue exclusion, MTT assay, and cell adhesion studies (for
cells treated in suspension) were conducted to confirm cell viability
after various treatments. For the trypan blue exclusion assay, cells
were stained with trypan blue, and the cells taking up the dye were
counted microscopically. For the MTT assay, we used a modified
colorimetric assay based on the selective ability of
living cells to reduce the yellow dye, MTT, to intracellular formazan
via mitochondrial enzymes, as described before.32 Briefly,
aliquots of cells treated for different times in suspension with the
various experimental agents were plated in multiwell plates, allowed to
attach, and subsequently treated with MTT (5 mg/mL) under standard
tissue-culture conditions. In monolayers treated with different agents,
MTT was added directly to the wells after treatment. Following
incubation for 4 hours, the supernatant was aspirated without
disruption of the formazan precipitate, and the intracellular formazan
crystals were dissolved by adding 150 µL of DMSO to each well. The
absorbance was measured at 570 nm using a microplate spectrophotometer.
For cell adhesion viability assays, aliquots of cells treated in
suspension were plated in multiwell plates, and the dead floating cells
remaining after 5 hours of incubation were counted under the
microscope. In all experiments, cells grown in medium and without any
treatment served as controls.
Statistics
All experiments were conducted in triplicate or quadruplicate
with 3 to 5 separate cultures. Data are presented as mean±SEM,
and statistical analysis was performed using ANOVA, paired
Student's t test or Fisher's least significant difference
test as appropriate. A value of P<0.05 was considered
statistically significant.
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Incorporation of cis-Parinaric Acid Into Smooth Muscle Cell Lipids
Figure 1A
shows a typical
high-performance liquid chromatogram of fluorescence
detected in phospholipids extracted from smooth muscle cells incubated
in the presence of hSA-cis-parinaric acid complex. Four
phospholipid peaks were well resolved and identified as
phosphatidylethanolamine, phosphatidylserine,
phosphatidylcholine, and sphingomyelin. Under the same excitation and
emission limits used above, fluorescent compounds were not
detected by HPLC in extracts from cells incubated with hSA in the
absence of cis-parinaric acid (data not shown).
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cis-Parinaric acid incorporation into different membrane
phospholipids in smooth muscle cells was dependent on the incubation
time of these cells with albumin-cis-parinaric acid
complex (Figure 2A). The maximal
incorporation of cis-parinaric acid into all detected
phospholipid classes was reached within 1 to 2 hours of incubation
(Figure 2A). cis-Parinaric acid was differentially
incorporated into the various phospholipids
(phosphatidylcholine>phos- phatidylethanolamine>phosphatidylserine>sphingomyelin).
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Effect of AMVN on Smooth Muscle Cell Membrane Lipids
Figure 1B
depicts a chromatogram of fluorescence
detected in cis-parinaric acid–acylated lipids extracted
from cis-parinaric acid–labeled smooth muscle cells
incubated in the presence of AMVN. Incubation of smooth muscle cells
with AMVN (500 µmol/L) induced significant oxidation of
cis-parinaric acid in all the detected phospholipids (Figure 2B) as indicated by the reduction in fluorescence. In
smooth muscle cells treated with 50 to 100 µmol/L of AMVN, no
oxidation of cis-parinaric acid–labeled phospholipids could
be observed (Figure 2B). Incubation of cis-parinaric
acid–labeled smooth muscle cells with 250 µmol/L AMVN resulted
in a decrease of cis-parinaric acid fluorescence of
phosphatidylserine, phosphatidylethanolamine, and
phosphatidylcholine by 39%, 22%, and 16%, respectively
(Figure 2B). The extent of oxidation of cis-parinaric
acid that was incorporated into the smooth muscle cell phospholipids
was dependent on the duration of incubation of these cells with AMVN
(data not shown). No loss in cell viability was observed in cells
treated for various times and concentrations of AMVN and as assessed by
trypan blue exclusion test, MTT assay, and cell adhesion assay (data
not shown).
Effect of Various Estrogens on the AMVN-Induced Oxidation of
cis-Parinaric Acid–Acylated Phospholipid in Smooth
Muscle Cells
2-Hydroxyestradiol, 4-hydroxytamoxifen, and PMC protected
cis-parinaric acid–acylated phospholipids against
AMVN-induced oxidation in a concentration-dependent manner, whereas
droloxifene and estrone did not (Figures 3 and 4).
The potency of PMC in protecting the various phos-pholipids was in the
following order:
phosphatidylethanolamine>phosphatidylserine>phosphatidylcholine
sphingomyelin.
Concentrations of 2-hydroxyestradiol and 4-hydroxytamoxifen higher than
250 ng/mL were as effective as PMC in preventing
AMVN-induced oxidations of phosphatidylethanolamine and more
effective than PMC in preventing
phosphatidylserine, phosphatidylcholine, and
sphingomyelin oxidation. However, at lower concentrations (<250
ng/mL), PMC was more effective than 2-hydroxyestradiol and
4-hydroxytamoxifen in protecting all the phospholipids (Figures 3 and 4). AMVN-induced oxidation of the various
phospholipids was completely blocked (100%) by 2.5 to 5 µg/L per
106 cells of PMC, 2-hydroxyestradiol, and
4-hydroxytamoxifen. The overall efficacy of the various estrogens and
phenolic compounds in protecting against lipid peroxidation was
PMC2-hydroxyestradiol>4-hydroxytamoxifen>droloxifeneestrone
(Figures 3 and 4).
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In smooth muscle cells pretreated for 20 hours,
physiological concentrations (0.3 ng/mL) of
2-hydroxyestradiol selectively decreased AMVN-induced peroxidation of
phosphatidylinositol and phosphatidylserine by 34%
and 25%, respectively (Figure 5). In
contrast to 2-hydroxyestradiol, estrone even at high concentrations (30
ng/mL) was a weak inhibitor of AMVN-induced peroxidation of
phosphatidylserine and phosphatidylinositol (Figure 5). Similarly to 2-hydroxyestradiol, 4-hydroxytamoxifen (40
ng/mL) and PMC (20 ng/mL) selectively protected against AMVN-induced
peroxidation of phosphatidylinositol and
phosphatidylserine (Figure 5), but not
phosphatidylethanolamine and phosphatidylcholine (data not shown). No
loss in cell viability was observed in cells treated for various times
and concentrations of the experimental agents and AMVN as assessed by
trypan blue exclusion test, MTT assay, and cell adhesion assay (data
not shown).
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Effect of AMVN on the Phospholipid Composition in Smooth
Muscle Cells
High-performance thin-layer
chromatographic analysis of total polar lipids is
shown in the Table. Phosphatidylcholine
and phosphatidylethanolamine represented the major fraction
of the total phospholipids in smooth muscle cells and was 50% and
27% of the total phospholipid content, respectively. Addi-tionally,
the other prominent phospholipids in order of their abundance were as
follows:
phosphatidylserine>phospha-tidylinositol>sphingomyelin>diphosphatidylglycerol>lyso-phosphatidylcholine>phosphatidic
acid. No significant differences in phospholipid distribution or
content were detected in smooth muscle cells following oxidative stress
imposed by incubation of the cells in the presence of AMVN
(Table), with the exception of a slight increase in
lysophosphatidylcholine, a relatively minor phospholipid.
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Effect of Various Estrogens on the AAPH-Induced Oxidation of
Intracellular Thiols and Glutathione
The incubation of smooth muscle cells with AMVN (500
µmol/L) for 2 hours at 37°C resulted in a mild oxidation (15% to
17%) of intracellular thiols (glutathione plus intracellular
thiols; data not shown). The GSH level, measured as thiol compound
after pretreatment of cells with glutathione-peroxidase and cumene
hydroperoxide in the presence of deferoxamine mesylate, was
23.81±6.34 nmol/106 cells. The intracellular
glutathione content in smooth muscle cells was 75% to 85% of total
thiol content in smooth muscle cells. The incubation of smooth muscle
cells with AAPH (20 mmol/L) for 2 hours at 37°C resulted in a
significant oxidation of the intracellular thiols and glutathione
(Figure 6, top panels). In the presence
of PMC, AAPH-induced oxidation of intracellular thiols was inhibited in
a concentration-dependent manner, suggesting that PMC protects
glutathione against oxidation. In contrast to PMC, 4-hydroxytamoxifen,
droloxifene, estrone, and 2-hydroxyestradiol increased the AAPH-induced
oxidation rate of thiols in smooth muscle cells. However, in the
absence of AAPH, these phenolic compounds did not affect the levels of
intracellular thiols and glutathione in the smooth muscle cells (data
not shown). Additionally, when pure glutathione was incubated in
phosphate buffer containing 100 µmol/L deferoxamine
mesylate, auto-oxidation of glutathione was not observed. However, when
AAPH (20 mmol/L) was added, the peroxyl radicals produced by AAPH
oxidized glutathione at a rate of 1.45 nmol/min. Moreover, PMC
protected glutathione from AAPH-induced oxidation in a
concentration-dependent manner (Figure 7B). In contrast to the protective
effects of PMC, the estrogens (4-hydroxytamoxifen, droloxifene,
estrone, and 2-hydroxyestradiol) did not inhibit AAPH-induced oxidation
of glutathione.
|
|
Effects of Various Estrogens on AMVN-Induced DNA Synthesis, Cell
Number, and Cell Migration
Treatment with AMVN stimulated DNA synthesis in a
concentration-dependent manner (P<0.001 versus 0.5% FCS;
Figure 7A). 2-Hydroxyestradiol and 4-hydroxytamoxifen inhibited
AMVN-induced DNA synthesis in a concentration-dependent manner. As
compared with 2-hydroxyestradiol, estrone was a very weak
inhibitor of AMVN-induced DNA synthesis (Figure 7B).
The lowest concentrations of estrone, 2-hydroxyestradiol, and
4-hydroxytamoxifen that inhibited AMVN-induced DNA synthesis were 0.3
ng/mL for 2-hydroxyestradiol, 30 ng/mL for estrone, and 0.4
ng/mL for 4-hydroxytamoxifen (Figure 7B). A 50% decrease in
AMVN-induced DNA synthesis in smooth muscle cells was observed at 3
ng/mL 2-hydroxyestradiol and 40 ng/mL 4-hydroxytamoxifen. In contrast
to 2-hydroxyestradiol, estrone at a concentration of 300 ng/mL
inhibited AMVN-induced DNA synthesis by only 10%. Similarly to the
effects on DNA synthesis, treatment with AMVN for 4 days increased cell
number, and these proliferative effects were significantly inhibited by
2-hydroxyestradiol and 4-hydroxytamoxifen, but not by estrone (Figure 8A).
|
FCS stimulated migration of smooth muscle cells, and this effect was
significantly enhanced in smooth muscle cells treated with AMVN (Figure 8B). In cells stimulated with FCS plus AMVN, 2-hydroxyestradiol
and 4-hydroxytamoxifen inhibited migration in a concentration-dependent
manner (Figure 8B), whereas estrone did not inhibit migration.
The lowest concentrations of 2-hydroxyestradiol and 4-hydroxytamoxifen
that inhibited cell migration was 0.3 ng/mL for 2-hydroxyestradiol and
0.4 ng/mL for 4-hydroxytamoxifen (Figure 8B).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The present study demonstrates that aortic smooth muscle cells labeled with cis-parinaric acid provide a sensitive and quantitative method to assess the effects of drugs on lipid peroxidation. Using this technique, this study establishes that AMVN-induced peroxidation of various smooth muscle cell membrane phospholipids is not prevented by estrone, an estrogen-receptor ligand, but is completely blocked by 2-hydroxyestradiol, a major metabolite of 17ß-estradiol that does not have a high affinity for the estrogen receptor.33 34 Moreover, 4-hydroxytamoxifen (a metabolite of tamoxifen with high affinity for estrogen receptors) effectively blocks peroxidation of smooth muscle cell phospholipids,8 while droloxifene, a tamoxifen analog known to be an estrogen-receptor ligand,35 does not prevent peroxidation. Both 2-hydroxyestradiol and 4-hydroxytamoxifen are as effective as PMC in protecting membrane phospholipids against peroxidation. Moreover, 2-hydroxyestradiol and 4-hydroxytamoxifen inhibit peroxyl radical–induced proliferation and migration of smooth muscle cells. In contrast to the effects on lipid peroxidation and cell growth, 2-hydroxyestradiol and 4-hydroxytamoxifen do not prevent AAPH-induced oxidation of cellular thiols/glutathione in smooth muscle cells. These studies provide the first evidence that the cardioprotective effects of estrogen and tamoxifen may in part be due to the direct antioxidant effects of their major metabolites on membrane phospholipids of smooth muscle cells.
Because our aim was to evaluate oxidative damage in cells membranes, we required a sensitive method for detecting oxidation of phospholipids. Thus, we selected the cis-parinaric acid/AMVN paradigm. In this regard, our results indicate that cis-parinaric acid covalently labels various phospholipids in smooth muscle cells in a differential and time-dependent fashion and that cis-parinaric acid fluorescence in the various phospholipids is uniformly reduced in smooth muscle cells treated with AMVN. These results indicate that smooth muscle cells can be labeled with cis-parinaric acid and that AMVN-derived peroxyl radicals cause peroxidation of a broad array of lipid components in the cell membrane. Even though cis-parinaric acid–labeled phospholipids constitute <1% of the total lipid content,36 we can detect AMVN-induced peroxidation in cis-parinaric acid–labeled smooth muscle cells, as well as protection against AMVN-induced peroxidation by PMC, a potent antioxidant. The actual content of various membrane phospholipids analyzed using conventional methods did not vary in control and AMVN-treated smooth muscle cells, indicating that decreases in unoxidized cis-parinaric acid in labeled smooth muscle cells were not due to loss of cis-parinaric acid–labeled phospholipids, but rather to the change in the oxidization state of incorporated cis-parinaric acid. Moreover, the finding that cell viability was not reduced in smooth muscle cells treated with AMVN suggests that the effects of AMVN on lipid peroxidation were not due to cell death. Together, these findings strongly suggest that cis-parinaric acid–labeled smooth muscle cells provide an excellent in vitro system to evaluate the effects of both oxidative stress-induced lipid peroxidation and the antioxidant capabilities of chemicals and new therapeutic drugs. In the present study, we found that AMVN-induced peroxidation of various membrane phospholipids and AMVN-induced cell growth are not prevented by estrone, but are completely blocked by 2-hydroxyestradiol. This finding suggests that the cardioprotective effects of 17ß-estradiol may, in part, be due to the direct antioxidant effects of its metabolite on smooth muscle cells. Differences in the antioxidant capabilities of estrone and 2-hydroxyestradiol may largely be due to the chemical nature of the molecule. Indeed, 2-hydroxyestradiol is a catechol estrogen and is a more potent antioxidant than 17ß-estradiol.6 33 It is also possible that, similar to 17ß-estradiol, the metabolism of estrone to 2-hydroxyestrone is necessary to induce its antioxidant effects. Indeed, a recent study has shown that 2-hydroxyestrone is several times more potent than estrone in protecting against lipid peroxidation.6 Moreover, as compared with 17ß-estradiol, 2-hydroxyestradiol is several times more potent in inhibiting DNA synthesis in smooth muscle cells37 and proliferation as well as extracellular matrix synthesis in cardiac fibroblast.38
Whether the cardioprotective effects of estrogens are receptor dependent is under intense debate. Recent studies suggest that the cardioprotective effects of estrogen(s) are non–receptor mediated39 ; however, the mechanisms involved remain unclear. Our finding that 4-hydroxytamoxifen, a phenolic metabolite of tamoxifen and an estrogen-receptor ligand, is as potent as 2-hydroxyestradiol in protecting phospholipids against AMVN-induced peroxidation and our finding that estrone and droloxifene, which bind to estrogen receptors with high affinity, are ineffective in preventing peroxidation, suggest that the non–receptor-mediated cardioprotective effects of estrogens may be due to direct antioxidant effects of their metabolites on smooth muscle cells. This notion is further supported by our observation that, similarly to 2-hydroxyestradiol, 4-hydroxytamoxifen inhibited AMVN-induced growth of smooth muscle cells. Nonetheless, further experiments will be required to determine whether the observed protective effects of estrogen and tamoxifen metabolites are greater in females and whether the effects are blocked by specific estrogen receptor antagonists.
To evaluate the antioxidant potency of various estrogens, we compared their effects with PMC, a homolog of the natural membrane antioxidant, and growth inhibitor vitamin E,40 41 a very potent antioxidant. An important observation in the present study is that 2-hydroxyestradiol and 4-hydroxytamoxifen are as effective as PMC in preventing lipid peroxidation. It is well documented that 4-hydroxytamoxifen is severalfold more potent than tamoxifen in preventing LDL oxidation, iron-induced microsomal peroxidation, and cardiovascular disease.8 Moreover, it has been suggested that the cardioprotective effects of tamoxifen are largely mediated via generation of 4-hydroxytamoxifen. This is in agreement with our observation that low concentrations (1 nmol/L) of 4-hydroxytamoxifen inhibited AMVN-induced growth of smooth muscle cells. With regard to 2-hydroxyestradiol, no studies have been conducted to compare its potency with that of 17ß-estradiol in inducing cardioprotective effects in vivo. However, numerous studies suggest that 2-hydroxyestradiol is the major metabolite of 17ß-estradiol and that some biological effects of estrogens are mediated in part through their metabolites.33 34 As compared with 17ß-estradiol and estrone, their 2-hydroxymetabolites (2-hydroxyestradiol and 2-hydroxyestrone) are severalfold more effective in preventing lipid peroxidation in liposomes.6 10 13 33 Hence, similarly to tamoxifen,8 estrogens may largely induce their cardioprotective effects via generation of the potent antioxidant metabolites. This hypothesis is further supported by our observation that low concentrations of 2-hydroxyestradiol, but not estrone, inhibited free radical–induced DNA synthesis in smooth muscle cells.
The cardioprotective effects of several drugs with antioxidant effects are thought to be mediated via modulation of the endogenous free radical scavenging system, including the glutathione reductase system.42 Hence, the possibility that estrone, 2-hydroxyestradiol, droloxifene, and 4-hydroxytamoxifen may also induce their cardioprotective effects by preventing peroxyl radical–induced oxidation of intracellular thiols in smooth muscle cells was also investigated. In smooth muscle cells treated with AAPH, the intracellular thiol/GSH is oxidized rapidly. In contrast to the effects on lipid peroxidation, 4-hydroxytamoxifen and 2-hydroxyestradiol did not prevent AAPH-induced oxidation of smooth muscle cell thiol/glutathione. Thus, the antioxidant effects of 2-hydroxyestradiol and 4-hydroxytamoxifen are exerted within the cell membrane, rather than in the cytoplasm.
Do estrogens prevent lipid peroxidation and free radical–induced growth in vivo? The observation that physiological concentrations (0.3 ng/mL) of 2-hydroxyestradiol inhibited free radical–induced growth and migration of smooth muscle cells suggests that 2-hydroxyestradiol may importantly contribute to the cardioprotective effects of estrogen in vivo. Moreover, the finding that pretreatment of cells for 20 hours with physiological concentrations of 2-hydroxyestradiol, but not estrone, selectively protects phosphatidylinositol and phosphatidylserine against AMVN-induced peroxidation suggests that estradiol metabolites may protect against peroxyl radical–induced growth via mechanisms linked to these phospholipids. In this regard, it is important to note that cell viability, as assessed by trypan blue exclusion, MTT assay, and a cell adhesion assay, was not reduced by prolonged treatment with AMVN, 2-hydroxyestradiol, 4-hydroxytamoxifen, or estrone. Therefore, cell death cannot account for the reduced AMVN-induced peroxidation by 2-hydroxyestradiol. Finally, it should be mentioned that plasma levels of 17ß-estradiol do not reflect the true physiological levels of antioxidant estrogens in the cell membrane, since in addition to 17ß-estradiol, several other estrogens and their metabolites are present in vivo,33 and estrogen metabolites concentrate in cell membranes.43 44 45
Regarding the mechanisms mediating oxidation-induced cell growth,
peroxidation of phospholipids has been shown to activate the
extracellular signal–regulated protein kinases (ERK1 and ERK2), induce
the expression of c-fos and c-jun oncogene
proteins, increase activator protein-1 DNA binding
activity, and induce nuclear factor-
ß.43 44 45 46 47 48
Moreover, the acidic membrane phospholipids phosphatidylinositol and
phosphatidylserine are known to selectively
activate protein kinase C activity, whereas
phosphatidylethanolamine and phosphatidylcholine do not.45
Moreover, oxidized metabolites of phosphatidylinositol have been shown
to induced cell migration. Hence, it is possible that
2-hydroxyestradiol and 4-hydroxytamoxifen prevent smooth muscle cell
growth by regulating protein kinase C activity by selectively
protecting peroxidation of phosphatidylinositol and
phosphatidylserine. However, further detailed
studies will be required to clarify the mechanisms by which
peroxidation of phospholipids enhances cell growth.
In conclusion, our findings suggest that 2-hydroxyestradiol and 4-hydroxytamoxifen may be more potent and clinically important than their parent compounds in preventing lipid peroxidation, growth of smooth muscle cells, and vaso-occlusive disorders.
|
Acknowledgments |
|---|
This work was supported by grants from the National Institutes of Health (HL 55314, HL 35909, and GM 47124), the Swiss National Science Foundation (32-54172.98), and in part by International Neurological Science Fellowship Program F05 NS10669, administered by National Institute of Neurological Disorders and Stroke, National Institutes of Health, in collaboration with Unit of Neuroscience, Division of Mental Health and Substance Abuse, World Health Organization.
Received July 28, 1998; accepted November 11, 1998.
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