Vol. 280, Issue 2, H876-H884, February 2001
Endothelial protective and antishock effects of a selective
estrogen receptor modulator in rats
Xin L.
Ma1,
Feng
Gao1,
Jun
Chen2,
Theodore A.
Christopher1,
Bernard L.
Lopez1,
Eliot H.
Ohlstein2, and
Tian-Li
Yue2
1 Division of Emergency Medicine, Department of Surgery,
Thomas Jefferson University, Philadelphia 19107; and
2 Department of Cardiovascular Pharmacology, SmithKline
Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406
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ABSTRACT |
This study investigated whether
idoxifene, a selective estrogen receptor modulator (SERM), exerted
protective effects against ischemia-reperfusion-induced shock.
Ovariectomized rats were treated with vehicle, idoxifene, or
17
-estradiol for 4 days. Rats were subjected to splanchnic artery
occlusion (SAO) followed by reperfusion (SOA/R). In vehicle-treated
rats, SAO/R resulted in hypotension, hemoconcentration, increased
plasma tumor necrosis factor (TNF)-
levels, intestinal neutrophil
accumulation, and endothelial dysfunction. 17-Estradiol treatment
increased plasma estradiol concentration and reduced SAO/R-induced
tissue injury. Idoxifene treatment had no effect on plasma estradiol
concentration but reduced SAO/R-induced hemoconcentration (+8.8 ± 1.3 vs. +14 ± 1.3% in the vehicle group, P < 0.01), TNF- production (98 ± 3.2 vs. 214 ± 13 pg/ml,
P < 0.01), and neutrophil accumulation (0.025 ± 0.005 vs. 0.047 ± 0.005 U/g protein, P < 0.01).
It also improved endothelial function, prolonged survival time
(172 ± 3.5 vs. 147 ± 8 min, P < 0.01), and
increased survival rate (69 vs. 23%, P < 0.01).
Moreover, treatment with 17-estradiol or idoxifene in vivo reduced
TNF--induced endothelial dysfunction in vitro. Taken together, these
results demonstrated that idoxifene exerted estrogen-like,
endothelial-protective, and antishock effects in ovariectomized rats,
suggesting that SERMs have therapeutic potential in tissue injury
resulting from ischemia-reperfusion.
ischemia-reperfusion; endothelium
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INTRODUCTION |
SPLANCHNIC ARTERY
OCCLUSION/REPERFUSION (SAO/R) shock is one of the most
severe types of circulatory shock. It is characterized by a marked
systemic decrease in postreperfusion blood pressure and is associated
with a high mortality rate (5, 26). Substantial evidence
indicates that endothelial dysfunction manifested as decreased
bioactive nitric oxide (NO) levels is one of the earliest pathophysiological changes occurring after ischemia-reperfusion (14) and that this early pathophysiological change
significantly contributes to subsequent functional and cellular injury
in a variety of pathophysiological pathways (18).
Endothelial dysfunction disturbs the balance between vasorelaxation and
vasoconstriction and thus may promote vasoconstriction and contribute
to the "no-reflow phenomena" seen after ischemia-reperfusion. It
may also exacerbate tissue injury indirectly by promoting neutrophil
[polymorphonuclear neutrophil (PMN)] accumulation, thus increasing
PMN-induced tissue injury. Treatment with free radical scavengers, NO
donors, and agents that preserve endothelial function have been shown
to exert significant antishock effects (4, 5, 26).
Estrogen replacement therapy after menopause has been shown to reduce
the morbidity and mortality of cardiovascular diseases (3). However, it is estimated that <10% of
postmenopausal women actually take estrogen replacement therapy to make
use of its apparent beneficial effects in preventing cardiovascular
diseases (13). The major reasons for this are fear of
estrogen-induced breast and uterine cancer (15). The
search for more acceptable and safer postmenopausal hormone replacement
therapies has led to the evaluation of compounds known as selective
estrogen receptor modulators (SERMs). A SERM is defined as a compound
that has estrogen agonism on one or more desired target tissues such as
the bone and liver, and estrogen antagonism and/or minimal estrogen
agonism in reproductive tissues such as the breast or uterus
(23). Several recent studies (10, 12) have
demonstrated that SERMs such as tamoxifen and raloxifene possess
similar antioxidant and vasorelaxation effects as estrogen. However,
whether or not SERMs may exert significant protective effects on
endothelial function associated with ischemia-reperfusion has not been
directly studied. Moreover, it was recently reported that
estrogen-treatment attenuated tumor necrosis factor (TNF)- production in rats subjected to SAO/R shock and prolonged survival time
in these animals. Whether or not a SERM may also possess similar
effects and protect tissue from SAO/R-induced injury has not been
evaluated. Accordingly, the aims of the present study were
1) to determine the dose-effect relationship of idoxifene, a
new SERM, on the severity of SAO/R-induced circulatory shock in
ovariectomized animals; and 2) to elucidate the potential
mechanisms by which idoxifene may exert its potential antishock effects.
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MATERIALS AND METHODS |
Female adult Sprague-Dawley rats (300-350 g) were obtained
from ACE Animals, and an ovariectomy was performed on all animals except those serving as sham-operated controls. Two weeks after ovariectomy, rats were randomly assigned to receive one of the following treatments for 4 days: 1) vehicle (0.1 M lactate
and 248 mM dextrose in saline) (27); 2)
idoxifene suspension (1 or 2 mg · kg
1 · day1, oral
gavage) (24); and 3) 17-estradiol suspension
(1 mg · kg1 · day1, oral
gavage; Sigma, St. Louis, MO) (24). The treatment interval was determined from a pilot study in which rats were treated with 1 mg · kg1 · day1 idoxifene
or 17-estradiol for 1 (single treatment 60 min before surgery) to 7 days (3-4 rats/group), and the effect of different treatments on
survival time was observed. The preliminary data demonstrated that
single-day treatment (60 min before surgical operation) accounted for
~40-50% of maximal protection. The protective effects of
idoxifene and 17-estradiol increased over the first 4 days of
treatment (including the last treatment 60 min before surgery) and
plateaued thereafter. Four days of treatment was thus chosen as an
optimal treatment duration. The experiments were performed in adherence
to NIH Guidelines on the Care and Use of Laboratory Animals
and were approved by the Thomas Jefferson University Committee on
Animal Care and Use.
Splanchnic ischemia-reperfusion injury.
On the fourth day after the start of treatment (60 min after the last
drug administration), rats were anesthetized with pentobarbital sodium
(50 mg/kg) via intraperitoneal injection. After anesthetization, the
trachea was cannulated with polyethylene (PE)-240 tubing to ensure a
patent airway. Polyethylene catheters (PE-50) filled with heparinized
0.9% NaCl solution were inserted into the left common carotid artery
for recording arterial blood pressure (ABP) using Cobe CDXIII
transducers (Lakewood, CO) and into the right external jugular vein for
supplemental pentobarbital injection to maintain a surgical plane of
anesthesia and for administration of drugs. After a midline laparotomy
was performed, the celiac and superior mesenteric arteries were
isolated from surrounding connective tissues near their aortic origins.
ABP signals were digitized via a MacLab data acquisition system (AD
Instruments, Milford, MA). Systolic blood pressure, diastolic blood
pressure, mean arterial blood pressure (MABP), and heart rate were
derived by computer algorithms.
Splanchnic ischemia-reperfusion was induced by total occlusion of the
superior mesenteric artery (SMA) and celiac artery with atraumatic
clamps for 60 min. Immediately before occlusion, the rats in all groups
were given heparin (250 U/kg iv) to prevent coagulation and ensure
reperfusion of the arteries 60 min later. After 60 min of ischemia, the
occlusive clamps were removed. The rats were then observed for an
additional 180 min or until the MABP fell to 45 mmHg. Survival time was
defined as the time elapsed from the removal of the occlusive clamps to
the time the MABP fell to 45 mmHg. Survivors were defined as rats
maintaining a MABP above 45 mmHg until 180 min after reperfusion. Sham
SAO/R rats were subjected to all the surgical procedures performed on SAO/R shock rats, including isolation of the SMA and celiac arteries, except that these arteries were not occluded.
At the end of surgery and at the end of the experiments, 0.1 ml of
arterial blood was drawn, and hematocrits (Hct) were determined with a
microhematocrit centrifuge (Marathon 6K; Fisher Scientific, Pittsburgh,
PA). To limit the influence of fluid replacement or supplementation
caused by catheter flushing on Hct readings and survival time, the
catheters were flushed hourly (at the end of ischemia and at 60 and 120 min of reperfusion) in every animal using 0.1 ml of heparinized 0.9%
NaCl (to prevent clot formation within the catheters). Therefore, every
animal received the same amount of fluid (0.3 ml) during the
experimental period.
Isolated SMA ring studies.
The SMA was isolated from all rats in each experimental group and
placed into ice-cold Krebs-Henseleit (K-H) buffer consisting of (in mM)
118 NaCl, 4.75 KCl, 2.54 CaCl2 · 2H2O,
1.19 KH2PO4, 1.19 MgSO4 · 7H2O, 25 NaHCO3, and
10.0 glucose. SMA segments were carefully cleaned of fat and loose
connective tissue and cut into two to three rings of 2-3 mm
length. These rings were then mounted on stainless steel hooks,
suspended and aerated (95% O2-5% CO2) in
7.5-ml K-H tissue baths at 37°C, and connected to FORT-10 force transducers (WPI, Sarasota, FL) to record changes via a MacLab data
acquisition system. The rings were then stretched to an optimum preload of 0.5 g of force, determined in previous experiments in
this laboratory (21), and allowed to equilibrate for 60 min. During this period, the K-H buffer in the tissue bath was replaced every 15 min, and the tension of the vascular rings was adjusted until
0.5 g of preload was maintained.
After equilibration, the rings were first exposed to a maximally
effective concentration (100 nM) of U-46619
(9,11-epoxymethano-PGH2; BioMol Research Laboratories,
Plymouth Meeting, PA) to ensure stabilization of the vascular smooth
muscle. The agonist was then washed out, and the rings were
reequilibrated. Twenty minutes after the initial washing, 50 nM U-46619
was added to each ring bath to generate ~0.5 g of developed force.
Once a stable contraction was obtained, acetylcholine (ACh), an agent
that induces vasorelaxation via stimulation of NO production from the
endothelium, was added to the bath in cumulative concentrations of
109-105 M to determine endothelial
function and agonist-stimulated NO production from the endothelium.
After the cumulative response stabilized, the rings were washed and
allowed to equilibrate to baseline. The procedure was then repeated
with an endothelium-independent vasodilator, acidified
NaNO2 (108-104 M), to
determine smooth muscle function. NaNO2 was prepared by dissolving the compound in 0.1 N HCl and titrating it to pH 2.0. Titrating distilled H2O to pH 2.0 and adding aliquots to
the bath did not produce any vasorelaxation.
Determination of tissue myeloperoxidase.
At the end of the experiments, intestinal tissue samples were obtained
for myeloperoxidase (MPO) determination. Small intestine MPO, an enzyme
occurring almost exclusively in neutrophils, was determined as
described previously (19). Briefly, the small intestine
(0.5-0.6 g) was homogenized in 0.5% hexadecyltrimethyl ammonium
bromide and dissolved in 50 mM of potassium phosphate buffer at pH 6.0 using a PRO 200 homogenizer (PRO Scientific, Monroe, CT). Homogenates
were centrifuged at 12,500 g at 4°C for 30 min. The
supernatants were then collected and reacted with 0.167 mg/ml
o-dianisidine dihydrochloride (Sigma) and 0.0005% H2O2 in 50 mM of phosphate buffer at pH 6.0. The change in absorbance was measured spectrophotometrically at 460 nm
(Beckman DU 640, Fullerton, CA). One unit of MPO was defined as that
quantity of enzyme hydrolyzing 1 mmol H2O2/min
at 25°C. The assays were performed without knowledge of the group
from which each sample originated.
Plasma estradiol assay.
Plasma estradiol was determined by radioimmunoassay by using a
double-antibody estradiol procedure following the manufacturer's manual (Diagnostic Products, Los Angeles, CA).
Plasma TNF- assay.
Plasma TNF- concentrations were determined using a rat TNF- ELISA
kit purchased from R&D System (Minneapolis, MN). In brief, 0.2 ml of
blood was withdrawn immediately before and at the end of reperfusion
(an arbitrarily chosen time point that may not reflect the peak value
of TNF- during shock) from five rats in each group (vehicle, 1 mg · kg1 · day1 idoxifene,
and 1 mg · kg1 · day1
17-estradiol). Blood was immediately centrifuged at 4°C, and plasma was stored at 
70°C for up to 1 wk. Plasma TNF-
concentrations were then assayed according to the method provided by
the company, and the results were expressed as picograms per milliliter.
Determination of ex vivo effects of 17-estradiol and idoxifene
on TNF--induced endothelial dysfunction in vitro.
In a separate study, we evaluated the ex vivo effects of idoxifene and
estradiol on TNF--induced endothelial dysfunction. Rats were treated
with either vehicle (n = 10) or drugs at their optimal
dose (1 mg · kg1 · day1
idoxifene, n = 10; 1 mg · kg1 · day1
estradiol, n = 10) for 4 days. The SMA was isolated
(without ischemia and reperfusion), and the vascular rings were
prepared in the same manner as described above. After an initial
determination of the vasorelaxation response to ACh, TNF- (10 ng/ml)
was added to each tissue bath, and rings were incubated with TNF-
for 2 h. After three complete washouts, the rings were again
checked for their endothelium-dependent and endothelium-independent
vasorelaxation. Differences between ACh-induced vasorelaxation before
and after TNF- incubation were calculated.
Statistical analysis.
Data were analyzed with the StatView or SuperANOVA programs (Abacus
Concepts, Berkeley, CA). MABP data were analyzed using two-way (time
and group) analysis of variance for repeated measures; post hoc testing
was done using the Tukey-Kramer high-significance difference
test. Hct, MPO, and vasorelaxation data were analyzed using
one-way ANOVA. Post hoc testing was done using the Bonferroni correction. Survival time was analyzed using the Kaplan-Meier estimation method followed by the Breslow-Gehan-Wilcoxon test. Survival
rate data were assessed by Fisher's exact probability test
(7). Probabilities of 
0.05 were considered to be
statistically significant.
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RESULTS |
Effects of 17-estradiol or idoxifene treatment on plasma
estradiol concentration.
Plasma estradiol concentration was significantly decreased in
ovariectomized rats when compared with sham-operated nonovariectomized rats (0.06 ± 0.01 vs. 0.21 ± 0.03 nM, P < 0.01, n = 9 rats/group). Administration of 1 mg · kg1 · day1
17-estradiol for 4 days increased plasma estradiol concentration to
2.8 ± 0.07 nM (n = 8, P < 0.01 vs. ovariectomized rats receiving vehicle). In contrast, administration
of 1 mg · kg1 · day1
idoxifene had no significant effect on plasma estradiol concentration (0.09 ± 0.03 nM, n = 8). These results
demonstrated that the 17-estradiol dose used in this study resulted
in pharmacological levels of plasma estradiol concentration that were
~13 times higher than those of normal female rats. Moreover, because
administration of idoxifene did not result in any significant change in
plasma estradiol level, the effect of idoxifene on
ischemia-reperfusion-induced injury was unlikely related to plasma
estradiol concentration.
Effects of idoxifene on severity of SAO/R-induced shock.
SAO/R results in a severe form of circulatory shock characterized by a
marked decrease in postreperfusion systemic ABP and an associated high
mortality rate. Figure 1 illustrates the
time course of MABP changes in the five groups of rats observed in this
study. The initial MABP in each group ranged from 90-100 mmHg and
were not statistically different. Moreover, the initial MABP of the
sham shock rats was 97 mmHg and did not vary significantly over the
course of the experiment, suggesting that the surgical procedures
performed did not contribute significantly to the severity of the SAO/R
injury state. All rats subjected to occlusion of their splanchnic
arteries developed a rapid rise in MABP of 20-30 mmHg followed by
a gradual return toward preocclusion levels during the 60 min of
occlusion.
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Fig. 1.
Effects of idoxifene (Ido) and 17-estradiol (Est) on mean
arterial blood pressure (MABP) in ovariectomized (Ovx) rats subjected
to sham splancic artery occlusion/reperfusion (SAO/R) or SAO/R. Number
after (or below) symbols indicates the number of surviving animals at
the indicated time point. I/R, ischemia-reperfusion; Is, ischemia; Re,
reperfusion. Numbers after Is or Re indicate the number of minutes of
ischemia or reperfusion, respectively. *P < 0.05, **P < 0.01 vs. Ovx + vehicle.
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MABP did not significantly differ among the four SAO/R groups during
the first 20 min of ischemia. However, those rats that were pretreated
with idoxifene at 1 mg · kg1 · day1 exhibited a
faster return of MABP toward the preocclusion level. Therefore, at 40 min and 60 min after occlusion, MABP in this idoxifene-treated group
was significantly lower than that in rats receiving only vehicle. All
rats subjected to SAO/R experienced an abrupt decrease (40-50
mmHg) in MABP on reperfusion of the splanchnic arteries followed by a
partial recovery and then a gradual secondary decline in MABP.
Pretreatment with idoxifene at either 1 or 2 mg · kg1 · day1
significantly attenuated the secondary decline in MABP. Specifically, at 180 min of reperfusion, MABP in these two groups was markedly higher
than that in vehicle-treated rats (P < 0.01).
Pretreatment with 17-estradiol at 1 mg · kg1 · day1 exerted
protective effects that were comparable with those exerted by idoxifene
at 1 mg · kg1 · day1 (Fig.
1).
All 10 sham SAO/R rats survived for the entire 3-h postreperfusion
observation period (survival rate 100%). The SAO/R rats receiving only
vehicle demonstrated a significantly shorter survival time (147 ± 8 min); only 3 of 13 rats survived at the end of the 3-h reperfusion
period (survival rate 23%). However, pretreatment with idoxifene
prolonged the survival time and increased the survival rate (Fig.
2). Pretreatment with 17-estradiol at
a dose of 1 mg · kg1 · day1
also significantly increased survival time and survival rate (165 ± 7 min and 70%, respectively).
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Fig. 2.
Effect of Ido and Est on survival time (A) and
survival rate (B) in Ovx rats subjected to sham SAO/R or
SAO/R. Bar heights represent mean values ± SE; numbers in bars
indicate number of rats in each experimental group. V, vehicle; Ido-1,
1 mg · kg1 · day1 Ido;
Ido-2, 2 mg · kg1 · day1
Ido; Est-1, 1 mg · kg1 · day1 Est.
*P < 0.05, **P < 0.01 vs. Ovx + vehicle.
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Effect of idoxifene and 17-estradiol on intravascular fluid
loss.
Hemoconcentration, resulting from the loss of fluid from the vascular
compartment due to altered microvascular function and increased
permeability, is a common pathophysiological change occurring in SAO/R
and significantly contributes to death in this animal model. There were
no significant differences in Hct readings among all groups of rats
studied before SAO/R (44 ± 1.1, 43 ± 1.0, 44 ± 1.7, 45 ± 1.3, and 43 ± 1.2% in five experimental groups). Hct
readings did not significantly change in sham SAO/R rats (from 44 ± 1.1 to 45 ± 1.3%, P > 0.05), indicating that
surgical procedures did not induce a significant increase in vascular
permeability. However, SAO/R rats receiving only vehicle exhibited a
marked increase in Hct reading (+14 ± 1.3%). Treatment with
either dose of idoxifene alleviated hemoconcentration (Hct increasing
after SAO/R: 8.8 ± 1.3 and 6.2 ± 1.7% in the two groups
receiving 1 or 2 mg · kg1 · day1 idoxifene,
P < 0.01 vs. vehicle). Pretreatment with
17-estradiol at 1 mg · kg1 · day1 also
resulted in a statistically significant attenuation of SAO/R-induced increase in Hct (7.9 ± 1.6%, P < 0.01 vs.
vehicle) (Fig. 3). These findings
indicate that, in this model of SAO/R injury, treatment with either
estrogen or a SERM curtailed an increase in Hct, an indirect
measurement of loss of fluid from the vascular compartment.
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Fig. 3.
Effect of Ido and Est on hematocrit (Hct) readings in Ovx rats
subjected to sham SAO/R or SAO/R. Bar heights represent mean
values ± SE; numbers in bars indicate number of rats in each
experimental group. **P < 0.01, ***P < 0.005 vs. Ovx + vehicle.
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Effects of idoxifene and 17-estradiol on endothelial function
after ischemia-reperfusion.
Endothelial dysfunction is one of the earliest pathophysiological
expressions occurring after organ ischemia and reperfusion. To clarify
whether a SERM protects the endothelium from ischemia-reperfusion injury, we studied the effects of idoxifene treatment on
endothelium-dependent vasorelaxation in isolated SMA segments. Figure
4 summarizes the maximal vasorelaxant
responses of isolated SMA rings from rats after SAO/R to an
endothelial-dependent vasodilator, ACh, or to an
endothelium-independent vasodilator, acidified NaNO2. SMA
rings from sham SAO/R rats exhibited complete vascular relaxation to both endothelium-dependent (105 M ACh) and the
endothelium-independent (104 M acidified
NaNO2) vasodilators. In contrast, the maximal
vasorelaxation to ACh was significantly decreased in the SMA rings from
vehicle-treated SAO/R rats (52 ± 4.3%). Treatment with either
dose of idoxifene (79 ± 3.7 and 72 ± 3.6%, respectively)
or 17-estradiol (74 ± 6.8%) significantly improved
vasorelaxation responses of SMA rings to ACh (Fig. 4A).
These results demonstrated that idoxifene, a new SERM, exerted a
significant estrogen-like protective effect on ACh-stimulated NO
release after ischemia and reperfusion.
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Fig. 4.
Effect of Ido and Est on endothelium-dependent (A) and
endothelium-independent (B) vasorelaxation in vascular rings
isolated from Ovx rats subjected to sham SAO/R or SAO/R.
**P < 0.01 vs. Ovx + vehicle. n = 18-20 rings from 10 to 15 rats/group.
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Because idoxifene and 17-estradiol treatment markedly
prolonged the survival time after SAO/R, the vascular segments from these animals were isolated after a longer period of reperfusion. It is
therefore possible that the enhanced vasorelaxation response to ACh
observed in drug-treated animals compared with vehicle-treated animals
was due simply to a prolonged reperfusion time in these animals. To
address this possibility, an additional series of experiments were
performed. Idoxifene- or 17-estradiol-treated rats (1 mg · kg1 · day1,
n = 8 rats/group) were exposed to SAO/R, and all
animals were euthanized at 147 min after reperfusion (mean survival
time for vehicle-treated rats). The maximal vasorelaxation response to ACh (86 ± 2.9 and 87 ± 3.3% respectively) in SMA rings
from idoxifene- and 17-estradiol-treated animals exposed to a
shorter reperfusion period was even greater than the vasorelaxation of
those rings from drug-treated animals euthanized at the end of
experiment. These results suggest that the endothelial protective
effects of idoxifene and 17-estradiol cannot be explained by a
longer period of reperfusion time.
To determine whether SAO/R may have altered the responsiveness of the
vascular smooth muscle to NO, we investigated the vasorelaxant effect
of acidified NaNO2 in SMA rings isolated from all seven groups. As summarized in Fig. 4B, acidified
NaNO2 induced a concentration-dependent vascular
relaxation, with full relaxation occurring at a NaNO2 concentration of 104 M. There were no significant
differences among any of the seven groups at any concentration of
NaNO2 tested.
Effect of idoxifene and 17-estradiol on neutrophil accumulation
in postischemic intestinal tissue.
It has been previously demonstrated that decreased NO production from
vascular endothelial cells is a triggering factor for neutrophil
adhesion to endothelial cells. It has also been shown that therapeutic
interventions that preserve endothelial NO production significantly
reduce neutrophil accumulation, which in turn attenuates postischemic
tissue injury. To determine whether SERM treatment, which results in
preservation of NO production, may also inhibit neutrophil
accumulation, we examined the effects of idoxifene treatment on MPO
activity in intestinal tissue. Figure 5
illustrates MPO activity of ileal tissue isolated from the five
experimental groups. All sham SAO/R rats exhibited normal-appearing
ileal tissue and low MPO activity (i.e., 0.003 ± 0.001 U/mg
protein), indicating that there was no significant neutrophil
accumulation in normal intestinal tissue. In contrast, the ileal MPO
activity in SAO/R rats receiving only vehicle was ~16 times higher
(0.047 ± 0.005 U/mg protein) than the MPO activity in the sham
SAO/R rat intestine (P < 0.01). Treatment with
idoxifene resulted in a significant decrease in MPO activity when
compared with rats treated with vehicle alone. Treatment with
17-estradiol also significantly reduced PMN accumulation in
ischemic-reperfused ileal tissue.
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Fig. 5.
Effect of Ido and Est on intestinal myeloperoxidase (MPO) activity
in Ovx rats subjected to sham SAO/R or SAO/R. Bar heights represent
mean values ± SE; numbers within bars indicate number of rats in
each experimental group. *P < 0.05, **P < 0.01, ***P < 0.005 vs. Ovx + vehicle.
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In those rats treated with idoxifene or 17-estradiol and euthanized
at a time point equivalent to the mean survival time of vehicle-treated
rats, ileal MPO activity (0.021 ± 0.004 and 0.019 ± 0.003 U/mg protein, respectively) was even slightly lower than that measured
in drug-treated animals killed at the end of the experiment (0.025 ± 0.005 and 0.028 ± 0.006 U/mg protein, respectively). These
results suggest that the lower MPO activity observed in idoxifene and
17-estradiol treated animals cannot be attributed to a longer period
of reperfusion time in these animals.
Effects of idoxifene on plasma TNF- accumulation.
TNF- is a cytokine that is implicated in the pathogenesis of
ischemic states, and exposure of endothelial cells to TNF- induces a
marked endothelial dysfunction. To further elucidate the mechanisms by
which a SERM may exert its endothelial protective effects, we measured
serum TNF- concentration. As illustrated in Fig.
6, SAO/R caused a marked increase in
serum TNF- concentration. Pretreatment with either idoxifene or
17-estradiol markedly attenuated the postreperfusion rise in TNF-
concentration.
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Fig. 6.
Effect of Ido and Est on postischemic serum tumor
necrosis factor (TNF)- elevation in Ovx rats. Bar heights represent
mean values ± SE; numbers in bars indicate number of rats in each
experimental group. *P < 0.05, **P < 0.01 vs. I/R + vehicle.
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Ex vivo effects of idoxifene and 17-estradiol on TNF--induced
endothelial dysfunction in vitro.
Consistent with previously reported results, incubation with TNF-
alone in isolated vascular segments significantly reduced vasorelaxation to ACh without affecting the vasorelaxation response to
NaNO2. In vivo treatment with either idoxifene or
17-estradiol markedly prevented this TNF--induced endothelial
dysfunction (Fig. 7). These results
suggest that in vivo treatment with a SERM may not only inhibit TNF-
production, as we have demonstrated in this study, but may also
directly block TNF--induced endothelial dysfunction even after
TNF- is released.
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Fig. 7.
Ex vivo effect of Ido and Est treatment on
TNF--induced endothelial dysfunction in vitro. **P < 0.01 vs. vehicle. n = 14 rings from 6 to 7 rats.
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DISCUSSION |
A previous experimental and clinical study (22) has
demonstrated that, in addition to its well-characterized beneficial effect on lipid profiles, estrogen may also exert cardiovascular protective effects via its favorable modulation of vasoreactivity. However, clinical application of estrogen in postmenopausal women is
greatly limited due to its reported stimulatory effect on reproductive tissues and increased risk of cancer in these organs. In the present study, we have evaluated the antishock and endothelial-protective effects of idoxifene, a new SERM that has previously been shown to
produce estrogen-like effects on bone and liver tissues and on plasma
lipid profiles but lacks estrogen effects or exert estrogen antagonist
effects on endometrial and breast tissues. We have demonstrated that in
a well-characterized ischemia-reperfusion-induced circulatory shock
model, pretreatment with idoxifene exerted significant protective
effects, as evidenced by increased MABP after reperfusion, prolonged
survival time, and increased survival rate. At 1 mg · kg1 · day1, a dose
that effectively prevents bone loss and lowers cholesterol level
without producing unwanted estrogenic effects on the endometrium (24), and without changing plasma estradiol concentration
in this study, the protective effects of idoxifene on all observed parameters were comparable to those of 17-estradiol. Increasing the
idoxifene dose to 2 mg · kg1 · day1 slightly
increased survival time and survival rate and further reduced
hemoconcentration and neutrophil accumulation. However, idoxifene at
this higher dose exerted slightly lesser protection against MABP
decline and endothelial dysfunction, although none of the differences
reached statistical significance compared with idoxifene at 1 mg · kg1 · day1. This may
be related in part to the ratio of agonist versus antagonistic effects
of SERMs. To our knowledge, this is the first study that directly
compared the cardiovascular protective effects of a SERM with
17-estradiol in an in vivo model that mimics a real
pathophysiological condition.
Idoxifene may exert its protective effects through several potential
mechanisms. Accumulating evidence now indicates that estrogen has a
direct effect on the vascular endothelium with increased NO bioactivity
(16, 22). Estrogen increases NO production via a
traditional genomic pathway that upregulates endothelial NO synthase
(NOS III) gene expression as well as a novel nongenomic pathway that
directly enhances NOS activity (16, 17). Estrogen may also
increase bioactive NO levels via inhibition of superoxide production
(2, 10), thus preventing NO from destruction by reactive
oxygen species. In our previous study (20) performed on
ovariectomized rats not subjected to ischemia and reperfusion, idoxifene restored basal NO release to a level that was not
significantly different from control female rats. In the present study,
we demonstrated that treatment with 17-estradiol as well as
idoxifene significantly increased bioactive NO levels in
ischemic-reperfused mesenteric vessels, as evidenced by increased
vasorelaxation to ACh, an endothelium-dependent vasodilator. Previous
studies from our laboratory and other investigators have demonstrated
that agents that preserve endothelial function or directly donate NO in
vivo exert significant antishock effects in this SAO/R shock model
(4, 6). By maintaining endothelial integrity and its NO
producing ability, idoxifene may thus improve postischemic tissue
perfusion and attenuate intravascular fluid loss. In our present study,
pretreatment with 17-estradiol as well as idoxifene significantly
attenuated Hct increase, an indirect index of intravascular fluid loss.
It is well known that PMNs play an important role in
ischemia-reperfusion-related tissue injury. PMNs can induce tissue
reperfusion injury by various mechanisms. First, activated PMNs release
a variety of cytotoxic substances, including proteases, collagenases, cytokines, leukotrienes, and cationic proteins, thereby causing tissue
damage (11). Second, adhered and aggregated PMNs can physically obstruct capillary flow and induce a no-reflow phenomena (11). This mechanical obstruction causes a regional
permanent ischemia and ultimately increases necrosis. Most importantly, a large body of evidence indicates that PMNs are the major source of
free radicals and thus are primarily responsible for free
radical-induced tissue injury after ischemia and reperfusion
(8). Our present study demonstrated that pretreatment with
17-estradiol as well as idoxifene markedly decreased MPO activity, a
reliable measurement of PMN accumulation in ischemic-reperfused tissue.
This anti-PMN activity of idoxifene may thus significantly contribute
to its cardiovascular protective effects.
Previous studies have demonstrated that cytokines, especially TNF-,
are important mediators that contribute to postischemic tissue injury
and ischemia-reperfusion-induced shock. Administration of human
recombinant TNF- produces a severe hypotension in dogs and a
decrease in vascular responsiveness to contractile agents in rats.
Moreover, accumulating evidence suggests that TNF- is involved in
postischemic endothelial dysfunction. TNF- has been shown to induce
a significant downregulation of NOS III (28). In cultured
endothelial cells, TNF- has been found to result in destabilization
of NOS III mRNA, possibly by inducing a protein that can enhance
degradation of mRNA and thus reducing transcription of NOS III
(1). In vivo infusion of lipopolysaccharide markedly inhibits endothelial NO production and ACh-induced vasodilatation (25). Moreover, a recent study (9) has
demonstrated that TNF- generated from smooth muscle cells in
response to interleukin-1 stimulation reduces NOS III expression in
a smooth muscle-endothelial cell coculture system. Therapeutic
interventions that reduce the production of cytokines, such as TNF-,
may thus decrease mRNA degradation and increase NOS III mRNA half-life,
thereby leading to preserved NO production.
To gain insight into the mechanism by which idoxifene may exert its
endothelial protective effect against ischemia-reperfusion injury, we
explored the possible involvement of TNF- in postischemic endothelial dysfunction and its protection by a SERM. Treatment with
idoxifene and 17-estradiol markedly attenuated TNF- release associated with splanchnic ischemia and reperfusion. This result is
consistent with that reported recently by Squadrito et al. (26). More importantly, we demonstrated for the first time
that in vivo administration of idoxifene and 17-estradiol markedly attenuated endothelial dysfunction resulting from in vitro TNF- incubation. Taken together, our results strongly suggest that idoxifene
may exert its endothelial protective effects and subsequent antishock
effects via its inhibitory effect on TNF- release in ischemic-reperfused tissue, thus reducing TNF--induced endothelial dysfunction and tissue injury. Moreover, our ex vivo results also suggest that idoxifene and 17-estradiol may directly block
TNF--induced downregulation of NOS in the endothelium.
In summary, we have demonstrated that idoxifene, a new SERM, produced
comparable antishock effects as that exerted by 17-estradiol. Preserved endothelial NO production, decreased PMN accumulation, and
attenuated TNF- production may all contribute to the observed protection from idoxifene. Further studies determining the mechanisms by which idoxifene may exert its endothelial protection, such as its
effects on peroxynitrite formation and on basal NO production after
ischemia and reperfusion, will provide further insight into the
antishock effects of idoxifene. Because SERMs such as idoxifene share
the beneficial effects of estrogen on lipid metabolism and vascular
endothelial function without adverse estrogenic effects on reproductive
tissues, they may prove to be a superior option over estrogen for
treatment and prevention of cardiovascular diseases.
| |
FOOTNOTES |
Address for reprint requests and other correspondence: X. L. Ma, Div. of Emergency Medicine, Jefferson Medical College,
1020 Sansom St., Philadelphia, PA 19107-5004 (E-mail:
Xin.Ma{at}mail.tju.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 July 2000; accepted in final form 5 October 2000.
| |
REFERENCES |
1.
Alonso, J,
Sanchez DM,
Monton M,
Casado S,
and
Lopez-Farre A.
Endothelial cytosolic proteins bind to the 3' untranslated region of endothelial nitric oxide synthase mRNA: regulation by tumor necrosis factor alpha.
Mol Cell Biol
17:
5719-5726,
1997[Abstract].
2.
Arnal, JF,
Clamens S,
Pechet C,
Negre-Salvayre A,
Allera C,
Girolami JP,
Salvayre R,
and
Bayard F.
Ethinylestradiol does not enhance the expression of nitric oxide synthase in bovine endothelial cells but increases the release of bioactive nitric oxide by inhibiting superoxide anion production.
Proc Natl Acad Sci USA
93:
4108-4113,
1996[Abstract/Free Full Text].
3.
Barrett-Connor, E,
and
Bush TL.
Estrogen and coronary heart disease in women.
JAMA
265:
1861-1867,
1991[Abstract/Free Full Text].
4.
Carey, C,
Siegfried MR,
Ma XL,
Weyrich AS,
and
Lefer AM.
Antishock and endothelial protective actions of a NO donor in mesenteric ischemia and reperfusion.
Circ Shock
38:
209-216,
1992[Web of Science][Medline].
5.
Christopher, TA,
Lopez BL,
Ma XL,
Feuerstein GZ,
Ruffolo RR, Jr,
and
Yue TL.
Effects of a hydroxylated metabolite of the beta-andrenoreceptor antagonist, carvedilol, on post-ischaemic splachnic tissue injury.
Br J Pharmacol
123:
292-298,
1998[Web of Science][Medline].
6.
Christopher, TA,
Lopez BL,
Yue TL,
Feuerstein GZ,
Ruffolo RR, Jr,
and
Ma XL.
Carvedilol, a new beta-adrenoreceptor blocker, vasodilator and free-radical scavenger, exerts an anti-shock and endothelial protective effect in rat splanchnic ischemia and reperfusion.
J Pharmacol Exp Ther
273:
64-71,
1995[Abstract/Free Full Text].
7.
Christopher, TA,
Ma XL,
Gauthier TW,
and
Lefer AM.
Beneficial actions of CP-0127, a novel bradykinin receptor antagonist, in murine traumatic shock.
Am J Physiol Heart Circ Physiol
266:
H867-H873,
1994[Abstract/Free Full Text].
8.
Das, DK,
and
Maulik N.
Antioxidant effectiveness in ischemia-reperfusion tissue injury.
Methods Enzymol
233:
601-610,
1994[Web of Science][Medline].
9.
De Frutos, T,
De Miguel LS,
Garcia-Duran M,
Gonzalez-Fernandez F,
Rodriguez-Feo JA,
Monton M,
Guerra J,
Farre J,
Casado S,
and
Lopez-Farre A.
NO from smooth muscle cells decreases NOS expression in endothelial cells: role of TNF-.
Am J Physiol Heart Circ Physiol
277:
H1317-H1325,
1999[Abstract/Free Full Text].
10.
Dubey, RK,
Tyurina YY,
Tyurin VA,
Gillespie DG,
Branch RA,
Jackson EK,
and
Kagan VE.
Estrogen and tamoxifen metabolites protect smooth muscle cell membrane phospholipids against peroxidation and inhibit cell growth.
Circ Res
84:
229-239,
1999[Abstract/Free Full Text].
11.
Duran, WN,
Silva MBJ,
and
Ferrante RJ.
Microcirculatory dysfunction in ischemia: reperfusion injury.
In: Reperfusion Injuries and Clinical Capillary Leak Syndrome, edited by Zikria BA,
Oz MO,
and Carlson RW.. Armonk, NY: Futura, 1994, p. 145-169.
12.
Figtree, GA,
Lu Y,
Webb CM,
and
Collins P.
Raloxifene acutely relaxes rabbit coronary arteries in vitro by an estrogen receptor-dependent and nitric oxide-dependent mechanism.
Circulation
100:
1095-1101,
1999[Abstract/Free Full Text].
13.
Harris, RB,
Laws A,
Reddy VM,
King A,
and
Haskell WL.
Are women using postmenopausal estrogens? A community survey.
Am J Public Health
80:
1266-1268,
1990[Abstract/Free Full Text].
14.
Harrison, DG.
Endothelial dysfunction in atherosclerosis.
Basic Res Cardiol
89, Suppl1:
87-102,
1994.
15.
Judd, HL,
Meldrum DR,
Deftos LJ,
and
Henderson BE.
Estrogen replacement therapy: indications and complications.
Ann Intern Med
98:
195-205,
1983.
16.
Kauser, K,
and
Rubanyi GM.
Potential cellular signaling mechanisms mediating upregulation of endothelial nitric oxide production by estrogen.
J Vasc Res
34:
229-236,
1997[Web of Science][Medline].
17.
Kim, HP,
Lee JY,
Jeong JK,
Bae SW,
Lee HK,
and
Jo I.
Nongenomic stimulation of nitric oxide release by estrogen is mediated by estrogen receptor alpha localized in caveolae.
Biochem Biophys Res Commun
263:
257-262,
1999[Web of Science][Medline].
18.
Lefer, AM,
Ma XL,
Weyrich A,
and
Lefer DJ.
Endothelial dysfunction and neutrophil adherence as critical events in the development of reperfusion injury.
Agents Actions Suppl
41:
127-135,
1993[Medline].
19.
Liu, GL,
Christopher TA,
Lopez BL,
Gao F,
Guo YP,
Gao E,
Knuettel A,
Feelisch M,
and
Ma XL.
SP/W-5186, a cysteine-containing nitric oxide donor, attenuates post-ischemic myocardial injury.
J Pharmacol Exp Ther
287:
527-537,
1998[Abstract/Free Full Text].
20.
Ma, XL,
Gao F,
Yao CL,
Chen J,
Lopez BL,
Christopher TA,
Disa J,
Gu JL,
Ohlstein EH,
and
Yue TL.
Nitric oxide stimulatory and endothelial protective effects of idoxifene, a selective estrogen receptor modulator, in the splanchnic artery of the ovariectomized rat.
J Pharmacol Exp Ther
295:
786-792,
2000[Abstract/Free Full Text].
21.
Ma, XL,
Lopez BL,
Christopher TA,
Birenbaum DS,
and
Vinten-Johansen J.
Exogenous NO inhibits basal NO release from vascular endothelium in vitro and in vivo.
Am J Physiol Heart Circ Physiol
271:
H2045-H2051,
1996[Abstract/Free Full Text].
22.
Miller, VM.
Gender, estrogen, and NOS: cautions about generalizations.
Circ Res
85:
979-981,
1999[Free Full Text].
23.
Mitlak, BH,
and
Cohen FJ.
Selective estrogen receptor modulators: a look ahead.
Drugs
57:
653-663,
1999[Web of Science][Medline].
24.
Nuttall, ME,
Bradbeer JN,
Stroup GB,
Nadeau DP,
Hoffman SJ,
Zhao H,
Rehm S,
and
Gowen M.
Idoxifene: a novel selective estrogen receptor modulator prevents bone loss and lowers cholesterol levels in ovariectomized rats and decreases uterine weight in intact rats.
Endocrinology
139:
5224-5234,
1998[Abstract/Free Full Text].
25.
Peters, TS,
and
Lewis SJ.
Lipopolysaccharide inhibits acetylcholine- and nitric oxide-mediated vasodilation in vivo.
J Pharmacol Exp Ther
279:
918-925,
1996[Abstract/Free Full Text].
26.
Squadrito, F,
Altavilla D,
Squadrito G,
Campo GM,
Arlotta M,
Arcoraci V,
Minutoli L,
Saitta A,
and
Caputi AP.
The involvement of tumour necrosis factor-alpha in the protective effects of 17 beta oestradiol in splanchnic ischaemia-reperfusion injury.
Br J Pharmacol
121:
1782-1788,
1997[Web of Science][Medline].
27.
Treinen, KA,
Rehm S,
and
Wier PJ.
An evaluation of the novel selective estrogen receptor modulator, idoxifene, for effects on reproduction in rats and rabbits.
Toxicol Sci
41:
199-207,
1998[Abstract/Free Full Text].
28.
Yoshizumi, M,
Perrella MA,
Burnett JC, Jr,
and
Lee ME.
Tumor necrosis factor downregulates an endothelial nitric oxide synthase mRNA by shortening its half-life.
Circ Res
73:
205-209,
1993[Abstract].
Am J Physiol Heart Circ Physiol 280(2):H876-H884
0363-6135/01 $5.00
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