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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 4 (August 15), 1999:
pp. 1330-1336
Increased Clearance Explains Lower Plasma Levels of Tissue-Type
Plasminogen Activator by Estradiol: Evidence for Potently Enhanced
Mannose Receptor Expression in Mice
By
Mirian Lansink,
Miek Jong,
Martin Bijsterbosch,
Marian Bekkers,
Karin Toet,
Louis Havekes,
Jef Emeis, and
Teake Kooistra
From the Gaubius Laboratory, TNO Prevention and Health, Leiden, The
Netherlands; the Division of Pharmaceutics, Leiden/Amsterdam Center for
Drug Research, University of Leiden, Sylvius Laboratories, Leiden, The
Netherlands; and the Department of Cardiology and Internal Medicine,
Leiden University Medical Center, Leiden, The Netherlands.
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ABSTRACT |
Several clinical studies have demonstrated an inverse relationship
between circulating levels of estrogen and tissue-type plasminogen
activator (t-PA). The present study was designed to test the hypothesis
that estrogens lower plasma levels of t-PA by increasing its clearance
from the bloodstream. 17 -Ethinyl estradiol (EE) treatment resulted
in a significant increase in the clearance rate of recombinant human
t-PA in mice (0.46 mL/min in treated mice v 0.32 mL/min in
controls; P < .01). The clearance of endogenous,
bradykinin-released t-PA in rats was also significantly increased after
EE treatment (area under the curve [AUC], 24.9 ng/mL · min in
treated animals v 31.9 ng/mL · min in controls; P < .05). Two distinct t-PA clearance systems exist in vivo: the low-density lipoprotein receptor-related protein (LRP) on
liver parenchymal cells and the mannose receptor on mainly liver
endothelial cells. Inhibition of LRP by intravenous injection of
receptor-associated protein (RAP) as a recombinant fusion protein
with Salmonella japonicum glutathione S-transferase (GST)
significantly retarded t-PA clearance in control mice (from 0.41 to
0.25 mL/min; n = 5, P < .001) and EE-treated mice (from
0.66 to 0.35 mL/min; n = 5, P < .005), but did
not eliminate the difference in clearance capacity between the 2 experimental groups. Similar results were obtained in mice in which LRP
was inhibited via overexpression of the RAP gene in liver by adenoviral
gene transduction. In contrast, administration of mannan, a mannose
receptor antagonist, resulted in identical clearances (0.22 mL/min in
controls and 0.24 mL/min in EE-treated mice). Northern blot analysis
showed a 6-fold increase in mannose receptor mRNA expression in the
nonparenchymal liver cells of EE-treated mice, whereas the parenchymal
LRP mRNA levels remained unchanged. These findings were confirmed at
the protein level by ligand blotting and Western blotting analysis. Our
results demonstrate that EE treatment results in increased plasma
clearance rate of t-PA via induction of the mannose receptor and could
explain for the inverse relationship between estrogen status and plasma t-PA concentrations as observed in humans.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE FIBRINOLYTIC SYSTEM, which is
responsible for the dissolution of fibrin in the circulation, requires
precise regulation to ensure that it is neither deficient nor
excessive. The physiological importance of the system in humans is
demonstrated by associations between impaired fibrinolysis and
thrombotic events on the one hand and between excessive fibrinolysis
and bleeding complications on the other. Clinical and epidemiological
studies suggest that lowered fibrinolytic activity, resulting in
enhanced fibrin deposition, is a significant contributor to the
development of atherothrombosis (for a review, see Emeis et
al1 and references therein).
Tissue-type plasminogen activator (t-PA) is a key enzyme in the
fibrinolytic process that converts the inactive proenzyme plasminogen
to plasmin, a broad-spectrum protease that cleaves fibrin. Human t-PA
is also of particular pharmacological interest because of its value in
the treatment of thromboembolic disorders.2 t-PA activity
in blood is regulated at several levels, including controlled synthesis
and release of t-PA from the vascular endothelium,3 the
presence of the physiological inhibitor plasminogen activator inhibitor
type-1 (PAI-1),4 and rapid hepatic clearance of
t-PA.5 It is well-documented that the synthesis of t-PA
(and PAI-1) is influenced by a variety of exogenous factors, such as
hormones, cytokines, growth factors, and vasoactive
compounds.6 Little is known about the regulation of hepatic
clearance capacity. Changes in t-PA clearance rate under various
physiological and experimental conditions are usually attributed to the
more rapid clearance of t-PA as compared with that of t-PA/PAI-1
complexes7 and/or to changes in the liver blood
flow.8
There are several indications of an inverse correlation between plasma
estradiol levels and plasma t-PA levels. Firstly, lower plasma
concentrations of t-PA are found in women as compared with men.9-11 Secondly, the use of estrogens by women on oral
contraceptives or hormone replacement significantly decreases plasma
t-PA levels.11-13 Thirdly, the administration of estradiol
(in combination with cyproterone acetate) to male-to-female transsexual
subjects markedly reduces circulating t-PA
concentrations.14 To estimate basal t-PA synthesis in the
transsexual subjects, a venous occlusion test was performed. No
significant difference in increment in venous plasma t-PA levels in
response to occlusion of the upper arm was found in the subjects before
and after hormone treatment (Emeis and Giltay, unpublished
observations), suggesting that estradiol lowers
circulating t-PA levels by increasing t-PA clearance rather than
altering t-PA synthesis. In line with this, in vitro studies using
cultured human vascular endothelial cells failed to demonstrate a
direct effect of estrogens on t-PA synthesis.15 In the
present communication, we report that 17 -ethinyl estradiol (EE)
treatment enhances the clearance rate of endogenous t-PA in rats and of
exogenously administered recombinant human t-PA in mice. Two major t-PA
receptors exist in liver, the low-density lipoprotein (LDL)
receptor-related protein (LRP) on predominantly liver parenchymal cells
and the mannose receptor mainly on liver endothelial
cells.5 By using specific receptor antagonists, we show
that the increase in clearance of t-PA in EE-treated mice occurs via
the mannose receptor. The role of the mannose receptor in the clearance
of t-PA is further substantiated by demonstrating an EE-increased
mannose receptor expression at the mRNA and protein level. These
results provide an explanation for the inverse relationship between
estradiol and plasma t-PA concentrations as observed in clinical studies.
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MATERIALS AND METHODS |
Materials.
Recombinant human t-PA expressed in Chinese hamster ovary cells
(Activase) was obtained from Genentech (San Francisco, CA). 17-Ethinyl estradiol, bradykinin, mannan, and peanut (arachis) oil
were purchased from Sigma Chemical Co (St Louis, MO). Hypnorm (10 mg/mL
fluanison and 0.315 mg/mL fentanyl citrate) was from Janssen
Pharmaceutica (Tilburg, The Netherlands) and Midazolam (5 mg/mL) from
Roche (Mijdrecht, The Netherlands). Collagenase (type IV) was purchased
from Boehringer Mannheim (Mannheim, Germany). The mouse monoclonal
human mannose receptor antibodies were prepared in our
laboratory.16 Enzyme immunoassay kits for determination of
human t-PA antigen (Thrombonostika t-PA) were obtained from Organon
Teknika (Boxtel, The Netherlands). Materials used in the rat t-PA
immunoassay (enzyme-linked immunosorbent assay [ELISA]) have been described previously.17 Receptor-associated
protein (RAP) was produced as a recombinant fusion protein with
Salmonella japonicum glutathione S-transferase (GST) and purified
according to a combination of the methods described by Herz et
al18 and Warshawsky et al.19 The RAP-GST
plasmid was kindly provided by Dr J. Herz (University of Texas, Dallas,
TX). Human trypsin-activated 2-macroglobulin was
prepared as described by Ziere et al.20 Other materials
used have been specified in the methods described or in the related references.
Preparation of recombinant adenoviral vectors.
The recombinant adenoviral vectors expressing either the RAP gene
(Ad-RAP) or the  -galactosidase gene (Ad--Gal) under the control
of cytomegalovirus (CMV) promoter were kindly provided by
Drs T. Willnow and J. Herz, respectively.21,22 The
generation of these vectors and the propagation and titration of the
recombinant adenovirus have been described previously.23
For in vivo adenovirus infection, the virus was subjected to 2 rounds
of purification by CsCl gradient centrifugation followed by extensive
dialysis against a buffer containing 25 mmol/L Tris-HCl, 137 mmol/L
NaCl, 5 mmol/L KCl, 0.73 mmol/L Na2HPO4, 0.9 mmol/L CaCl2, 0.5 mmol/L MgCl2, pH 7.45, at
4°C. After dialysis, mouse serum albumin was added to 0.2%
(wt/vol) and glycerol to 10% (vol/vol), and aliquots of the virus
stocks were frozen in liquid N2 and stored at
 80°C. Routinely, virus titers of the stocks varied from 1 to
5 × 1010 plaque-forming units (PFU)/mL. PFU (3 × 109) in a total volume of 200 µL (diluted with
phosphate-buffered saline [PBS]) were injected into the tail vein of
LDL receptor (LDLR)-deficient mice. t-PA clearance studies were
performed at day 5 after virus injection.
Animals.
C57 black6/B6 mice and Wistar rats were obtained from Iffa-Credo
(Someren, The Netherlands). LDLR-deficient mice were purchased from the
Jackson Laboratory (Bar Harbor, ME). For experiments, male rats (8 to
12 weeks old) and male mice (8 to 14 weeks old) were used. The animals
were housed as an experimental group with a 12-hour light cycle and
free access to drinking water and standard (chow) diet. All
experimental procedures were performed in accordance with The
Netherlands law on experiments with animals.
Estradiol treatment.
Rats and mice were injected 4 times subcutaneously with EE in arachis
oil (5 mg/kg body weight/day), either during 4 consecutive days or on
days 1, 4, 5, and 6. The LDLR-deficient mice (which were used in
parallel for very low-density lipoprotein clearance studies) received 3 subcutaneous injections of 100 µg EE in arachis oil (1 injection
every 2 weeks); control animals received arachis oil only. Experiments
were always performed 1 day after the last estradiol injection in case
of the short-term treatment or 2 weeks after the third estradiol
injection in case of the long-term treatment of LDLR-deficient mice. No
differences in t-PA clearance characteristics were observed for the
various EE treatment protocols.
Clearance of bradykinin-released t-PA in rats.
EE- or vehicle-treated rats were anesthetized with intraperitoneal
Nembutal (60 mg/kg body weight) and cannulated, and bradykinin (50 µg/kg body weight) was injected as a bolus into the vein of the
penis. Blood was collected immediately before and at different time
intervals (1 to 10 minutes) after bradykinin injection through a
carotid artery cannula, and citrated plasma was prepared. Rat t-PA
antigen concentrations were determined in citrated plasma by
ELISA.17
Plasma clearance of exogenous t-PA in mice; effect of inhibitors.
EE- or vehicle-treated mice were anesthetized by injection of 60 µL
Hypnorm/30 g body weight and 40 µL Midazolam/30 g body weight.
Fifteen micrograms of recombinant human t-PA in 200 µL sterile saline
was injected into a tail vein. Blood (35 to 40 µL) was collected
immediately after t-PA injection and at different time intervals (1 to
10 minutes) thereafter, and citrated plasma was prepared. Human t-PA
antigen levels were determined in citrated plasma by ELISA. In studies
in which mannan (5 mg/kg body weight) was administered, competitor was
injected 1 to 3 minutes before administration of t-PA. RAP-GST (40 mg/kg body weight) was preinjected 1 minute before t-PA injection. In
case of Ad-RAP or Ad--Gal infections, LDLR-deficient mice were
injected with 3 × 109 PFU of virus in a total volume
of 200 µL (diluted with PBS) into a tail vein, 5 days before the t-PA
clearance studies. At the end of experiments, livers were rapidly
removed and immediately frozen in liquid nitrogen for preparation of
total RNA or membrane fragments.
Isolation of liver cells.
Mouse parenchymal and nonparenchymal liver cells were isolated by a
procedure similar to one developed for rats.24 In short, the liver was preperfused for 8 minutes with Ca2+-free
Hanks' buffer, followed by a 8 minutes perfusion with Hanks' buffer
containing 0.05% (wt/vol) collagenase (flow rate, 14 mL/min). The
resulting cell suspension was filtered, and parenchymal and nonparenchymal cells were subsequently separated by differential centrifugation and density gradient centrifugation as described in
detail earlier.24
Isolation of total RNA and Northern blot analysis.
RNA from liver was prepared following the procedure of Chomczynski and
Sacchi.25 In short, frozen liver samples were triturated in
liquid nitrogen in a mortar, resuspended at 100 mg/mL in guanidinium thiocyanate/phenol-chloroform extraction buffer, and homogenized mechanically using a motor-driven Potter-Elvehjem homogenizer (10 strokes at 0°C). Total RNA was isolated and electrophoresed in a
1% (wt/vol) agarose gel under denaturing conditions using 1 mol/L
formaldehyde, blotted, and hybridized as described
previously.26 The following cDNA fragments were used as
probes in the hybridization experiments: a 6-kb Xho
I-EcoRI fragment of the human LRP cDNA,27 a 5.1-kb
full-length EcoRI fragment of the mouse mannose
receptor28 (kindly provided by Dr R. Ezekowitz, Harvard
University, Boston, MA), and a 1.2-kb Pst I fragment of a rat
glyceraldehyde-3-phosphate dehyrogenase (GAPDH) cDNA provided by Dr R. Offringa (Leiden University, Leiden, The Netherlands).
Preparation of membrane fragments and ligand-blotting analysis.
Frozen liver samples were triturated in liquid nitrogen in a mortar and
solubilized (at 60 mg/mL) in PBS containing 0.05% (vol/vol) Tween 20. After homogenizing using a motor-driven Potter-Elvehjem homogeniser (10 strokes at 0°C), debris and nuclei were removed by centrifugation
at 1,000g for 15 minutes. The supernatants containing the
membrane fragments were stored at 20°C until use. Samples (6 µL) were resolved by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) in a 5% gel using an SDS-concentration of
0.1% (wt/vol). After electrophoresis, the proteins were transferred electophoretically to Protan nitrocellulose filters (Schleicher and
Schuell, Dassel, Germany) in a buffer of 25 mmol/L Tris-HCl, 190 mmol/L
glycine, pH 8.6, 20% (vol/vol) methanol, and 0.1% (wt/vol) SDS at 1 mA/cm2 overnight, using a Pharmacia-LKB semi-dryblott
apparatus (Pharmacia-LKB, Uppsala, Sweden). The filters
were blocked with 10 mmol/L Tris-HCl, pH 8.0, containing 150 mmol/L
NaCl, 1% (wt/vol) skim milk, and 0.1% (vol/vol) Tween-20 for 1.5 hours, followed by incubation with 125I-GST-RAP at a
concentration of 2 nmol/L in 10 mmol/L Tris-HCl, pH 8.0, 150 mmol/L
NaCl, 0.5% (wt/vol) skim milk, and 0.1% (vol/vol) Tween-20 at room
temperature for 3 hours. After incubation, the filters were washed
extensively with 30 mL 10 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, and
0.1% (vol/vol) Tween-20 for 3 hours. Bound ligand was visualized by autoradiography.
Western blotting.
For Western blotting, samples (7.5 µL) of membrane fragments were
diluted 1 to 1 in Laemmli incubation buffer (50 mmol/L Tris-HCl pH 6.8, 1% [wt/vol] SDS, 10% [vol/vol] glycerol, and 8 mol/L urea) and
separated on a 5% to 18% (wt/vol) Laemmli gel. After electrophoresis, the proteins were transferred to Protan nitrocellulose in a buffer of
192 mmol/L glycine, 25 mmol/L Tris-HCl (pH 8.3), and 10% (vol/vol) methanol at 300 mA/cm2 overnight using a wet-blot apparatus
(Hoefer Scientific Instruments, San Francisco, CA). The filters were
blocked with 1% (wt/vol) skim milk in buffer A consisting of 0.5 mol/L
NaCl, 20 mmol/L Tris-HCl (pH 7.5), and 0.05% (vol/vol) Tween 20 for
0.5 hour at room temperature, followed by incubation with a mouse
mannose receptor monoclonal antibody (MoAb 15.2)16 at a
concentration of 1 µg/mL in buffer A for 1.5 hours at room
temperature. Next, the blots were washed 3 times with buffer A and
incubated for 1.5 hour at room temperature with rabbit antimouse RaM-PO
(1:5,000; Nordic, Tilburg, The Netherlands) as a conjugate. Finally,
the blots were stained with the peroxidase substrate BM-blue
(Boehringer Mannheim, Mannheim, Germany).
Statistics.
The unpaired Student's t-test was used to determine
statistical significance of the values obtained.
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RESULTS |
Effect of estrogen treatment on plasma clearance of t-PA in rats and
mice.
To evaluate whether estrogen treatment influences the clearance of
t-PA, we pursued 2 approaches. Firstly, we determined the disappearance
of exogenous recombinant human t-PA in EE-treated and control mice. As
shown in Fig 1A for a bolus injection of 15 µg of t-PA, clearance of t-PA was significantly faster in EE-treated mice than in control mice (0.47 ± 0.04 mL/min
v 0.33 ± 0.04 mL/min, respectively; n = 5, P < .01). Secondly, we determined the effect of EE on the clearance
of endogenous, bradykinin-released t-PA.29,30 For
practical reasons of blood sampling, these studies were performed in
rats (Fig 1B). At baseline, before the infusion of bradykinin, plasma
t-PA antigen levels were 2.0 ± 0.14 ng/mL and 2.5 ± 0.04 ng/mL
in EE-treated and control rats, respectively (n = 6, P < .05). Plasma t-PA antigen levels peaked in both experimental groups 1 minute after bradykinin injection and peak levels were not
significantly different in the EE-treated rats (8.7 ng/mL) versus the
control rats (8.4 ng/mL), indicating that bradykinin had induced
similar amounts of t-PA in both groups. t-PA antigen subsequently
decreased faster in the EE-treated group than in the control group
(area under the curve [AUC] of 24.9 ± 3.4 ng/mL · min and 31.9 ± 2.6 ng/mL · min, respectively; P < .05). Analysis of
the clearance curve showed that release of t-PA was completed at 1 minute. On the assumption that the biosynthesis of t-PA is unaffected,
we can calculate from the observed change in clearance and a plasma t-PA value in control rats of 2.5 ± 0.04 ng/mL a steady-state plasma t-PA concentration after estradiol treatment of 1.95 ± 0.23 ng/mL as compared with an experimentally determined value of 2.0 ± 0.14 ng/mL in EE-treated rats. The observed change in plasma t-PA
clearance therefore fully explains the observed decrease in plasma t-PA
concentration after EE treatment of the rats.
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| Fig 1.
Effect of EE treatment on plasma clearance of exogenous
human t-PA in mice and endogenous, bradykinin-released t-PA in rats. As
described in Materials and Methods, mice received injections into a
tail vein with 15 µg of recombinant human t-PA (A) or rats received
injections into a tail vein with 50 µg/kg body weight bradykinin (B),
after pretreatment of the animals with EE or vehicle. Blood samples
were collected at the indicated times and t-PA antigen
concentrations were determined by ELISA. Data points are the mean ± SD and represent 1 of 3 similar experiments performed in 5-fold (A) and
1 experiment performed in triplicate (B). ( ) Vehicle; ( ) EE.
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Effect of RAP and mannan on the plasma clearance of t-PA in mice.
The clearance of t-PA from the circulation is mainly mediated via 2 independent receptor systems, the LRP and the mannose receptor. To gain
more insight into the receptor system responsible for the EE-induced
increase in t-PA clearance, we blocked uptake of t-PA by each of the 2 receptor systems by specific antagonists.
Preinjection of GST-RAP (40 mg/kg), a dose previously shown to block
t-PA clearance via LRP in rats,31 reduced t-PA clearance in
control mice (from 0.41 ± 0.02 mL/min to 0.25 ± 0.02 mL/min, n = 4) and in EE-treated mice (from 0.66 ± 0.08 mL/min to 0.35 ± 0.05 mL/min, n = 5), but the difference in clearance of t-PA between
the 2 experimental groups remained maintained
(Fig 2A). Because GST-RAP is very rapidly
cleared from the blood circulation,20 we also evaluated the
functional effect of RAP that was overexpressed in the liver of mice
using an adenoviral gene transfer technique.32 As a control
for LRP blockade, we measured the clearance of radiolabeled 2-macroglobulin, a specific ligand for LRP. In
accordance with the data shown by Narita et al,32 5 days
after injection with AdCMV-RAP, mice were unable to clear
2-macroglobulin from the circulation, whereas the
control, Ad-LacZ-infected animals rapidly cleared this ligand for LRP
(data not shown). Also, in the Ad-RAP-infected mice clearance of t-PA
was increased in EE-treated mice as compared with control animals (0.24 ± 0.04 mL/min v 0.14 ± 0.02 mL/min, n = 3, P < .01; Fig 3).
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| Fig 2.
Effect of GST-RAP and mannan on the plasma clearance of
t-PA in control and EE-treated mice. As described in Materials and
Methods, EE- or vehicle-treated mice received injections into a tail
vein with 15 µg of recombinant human t-PA, 1 minute after
preadministration of 40 mg/kg body weight GST-RAP or PBS (A) or 1 to 3 minutes after preadministration of mannan or PBS (B). Blood samples
were collected at the indicated times and t-PA antigen concentrations
were determined by ELISA. Data points are the mean ± SD of 4 or 5 mice in each treatment group. (A) () Vehicle; () EE; ( )
vehicle and GST-RAP; ( ) EE and GST-RAP. (B) () Vehicle; () EE;
() vehicle and mannan; () EE and mannan.
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| Fig 3.
Effect of preadministration of AdCMV-RAP on plasma
clearance of t-PA in control and EE-treated mice. As described in
Materials and Methods, EE- and vehicle-treated mice were injected with
AdCMV-RAP or AdLacZ. Five days after virus administration, mice were
injected with 15 µg recombinant human t-PA. Blood samples were
collected at the indicated times and plasma t-PA concentrations were
determined by ELISA. Data points are the mean ± SD of 3 mice in each treatment group. () Vehicle and AdCMV-RAP; () EE and
AdCMV-RAP; () vehicle and AdLacZ; () EE and AdLacZ.
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To examine the contribution of the mannose receptor to the EE-induced
t-PA clearance, we blocked this receptor system by preinjection of mice
with mannan, a mannose receptor ligand. Preadministration of mannan (5 mg/kg), a dose previously shown to block clearance of ovalbumin, a
mannose-terminated glycoprotein, reduced plasma clearance of t-PA from
0.33 ± 0.04 mL/min to 0.22 ± 0.01 mL/min in control mice and
from 0.47 ± 0.04 mL/min to 0.25 ± 0.02 mL/min in EE-treated
mice (Fig 2B). Thus, in contrast to RAP-blockade of LRP,
preadministration of mannan abolished the faster clearance of t-PA in
EE-treated animals (Fig 2B). These results indicate that the
EE-increased t-PA clearance is mediated via the mannose receptor system.
Effect of EE-treatment on hepatic mannose receptor and LRP mRNA
levels.
Consistent with earlier observations in rats,33 we found
hepatic LRP mRNA expression in mice almost exclusively in hepatocytes, whereas the mannose receptor mRNA was demonstrated mainly in the nonhepatocyte (Kupffer cell/endothelial cell) fraction (data not shown). To further substantiate the EE-induced mannose receptor capacity, we performed Northern blotting studies to compare mannose receptor mRNA expression in livers from control and EE-treated mice. EE
treatment strongly increased liver mannose receptor levels (6.1- ± 1.1-fold, n = 3, P < .05), but not LRP mRNA levels (1.1- ± 0.1-fold induction, n = 3, not significant;
Fig 4). These effects of EE treatment on
the gene level were confirmed at the protein level. As shown in
Fig 5, Western blotting showed an increase in 180-kD mannose receptor protein signal in a liver membrane preparation of EE-treated animals as compared with that of control animals. The amount of 600-kD LRP protein, as determined by ligand blotting with 125I RAP, showed no significant difference
between the two groups.
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| Fig 4.
Effect of EE treatment on hepatic LRP and mannose
receptor mRNA expression in mice. (A) Total RNA was extracted from
livers from vehicle- (lane 1) or EE-treated mice (lane 2) and analyzed
by Northern blotting for LRP and mannose receptor (MR) mRNA levels. As
a control for equal loading, the blots were probed with the cDNA for
GAPDH mRNA. (B) The signals for LRP and MR mRNA were quantified by
phosphoimager analysis and adjusted for the corresponding GAPDH mRNA
signals. The results shown are the amounts of hepatic LRP and MR mRNA
in EE-treated mice relative to those found in vehicle-treated mice.
Data are expressed as the mean ± SD of 3 independent experiments
consisting of at least 4 animals in each treatment group.
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| Fig 5.
LRP and mannose receptor expression in livers from
control and EE-treated mice. Membrane fragments were isolated from
livers of mice treated for 4 days with vehicle () or EE. Solubilized
membrane proteins were subjected to SDS-PAGE and transferred to a
nitrocellulose membrane, as described in Materials and Methods. LRP (A)
was visualized by incubating the blot with 125I GST-RAP. MR
(B) was incubated with an MoAb against the MR and visualized as
described in Materials and Methods. Equal loading was controlled for by
Ponceau coloring of the blots.
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DISCUSSION |
The present study demonstrates that EE administration significantly
increases the clearance of endogenous, bradykinin-released t-PA in rats
and that of exogenously administered recombinant human t-PA in mice.
This increased t-PA clearance after EE treatment is most likely the
result of an increase in mannose receptor-mediated clearance. Firstly,
blocking of the LRP either by injection of GST-RAP or by overexpression
of RAP by adenoviral gene transduction did not eliminate the difference
in clearance between control and EE-treated mice. Secondly, in the
presence of the mannose receptor antagonist mannan, t-PA clearance in
control and EE-treated mice became identical. Thirdly, EE treatment of
mice induced a 6-fold increase in liver mannose-receptor mRNA
expression, whereas the amount of LRP mRNA in the EE-treated animals
did not differ from that in control animals. These findings were
confirmed at the protein level by Western blotting and ligand blotting.
Although the experiments were all performed in mice and rats, the
results are likely to be relevant to observed changes in t-PA levels
after estrogen administration in humans and also to observed gender
differences in plasma t-PA concentrations because of the similarities
between rodent and human t-PA clearance systems. In both rodents and
humans, the main clearance organ of t-PA is the liver.34,35
Also, the distribution of LRP and mannose receptor over the different
liver cell types is similar in rodents and humans. Both in human liver
and rat liver, LRP is mainly on hepatocytes, whereas the mannose
receptor is present on liver endothelial cells and Kupffer
cells.36-38 Our data in mice are in line with this, showing
expression of LRP in hepatocytes and of mannose receptor in
nonparenchymal cells.
An increased clearance of t-PA by estrogens as found in this study
explains, at least in part, the inverse association between plasma t-PA
levels and estrogens as observed in several clinical studies. In a
cross-sectional study, t-PA (and PAI-1) antigen levels were lower in
premenopausal women than in age-matched men, with the sex difference
disappearing after menopause.11 t-PA and PAI-1 antigen
levels were significantly higher in postmenopausal than in
premenopausal women.11,39 Studies on the use of oral contraceptives,12,13 hormone replacement
therapy,11 or studies with male-to-female
transsexuals14 also show an inverse association between
plasma t-PA concentrations and estrogen. The possibility that estrogens
decrease plasma t-PA levels directly by decreasing endothelial t-PA
production is not very likely. As a follow-up of our study with
male-to-female transsexual subjects,14 we performed venous
occlusion tests in a very similar group of subjects to estimate t-PA
synthesis before and after hormone treatment. Whereas a decrease in
basal plasma t-PA levels after estrogen treatment was confirmed, no
significant difference in increment in venous plasma t-PA levels in
response to occlusion of the upper arm was found (Emeis and Giltay,
manuscript submitted). In accordance with this finding, in vitro
studies with cultured human endothelial cells have shown that EE and
17- estradiol do not influence t-PA synthesis.15
However, we cannot exclude the possibility that other mechanisms also
contribute to the decreased t-PA antigen levels after estrogen
treatment. For instance, estrogen administration is usually associated
with lower plasma PAI-1 levels.13,14,40,41 As a
consequence, less t-PA/PAI-1 complex is formed, which may result in
faster clearance of unbound t-PA.7
Our finding that estrogens increase mannose receptor expression is able
to explain the lower levels of other mannose receptor ligands in women
as compared with men, like -glucuronidase and N-acetyl-
glucosaminidase.42,43 An effect of estrogen on the concentration of blood components via altered clearance is not unique
for t-PA. Treatment of rats with EE leads to a marked increase in the
number of LDL-receptors, resulting in lower plasma LDL-cholesterol levels as a result of increased clearance.44
Although the main point in this study is that upregulation of the
mannose receptor in nonparenchymal liver cells explains increased t-PA
clearance after estrogen administration, this effect of estrogen on
mannose receptor expression may also have consequences for other cell
types that express the mannose receptor, including tissue macrophages,
dendritic cells, and sperm cells, and thus may have more general
physiological importance also. In addition to the clearance of
glycoproteins, the mannose receptor functions to mediate the
phagocytosis of infectious agents, participates in antigen
presentation, is involved in the homing of lymphocytes to the spleen,
and plays a role in sperm fertility, and recent advances include
evidence that the mannose receptor is associated with a signal
transduction pathway leading to cytokine production.5,45 An
increase in mannose receptor expression by estrogens may impact on
these functions. However, it is uncertain whether the regulation of
expression of the mannose receptor in other cell types in similar to
that in nonparenchymal liver cells.5
The molecular mechanism underlying the EE-induced mannose-receptor
expression in mice was not the subject of the present study, but most
probably involves estrogen receptor-mediated stimulation of
mannose-receptor gene transcription. The recent description of a second
estrogen receptor, named ER,46,47 brings up many questions regarding the possibly distinct biological roles for the 2 estrogen receptor subtypes ( and ), their tissue distribution, and their ligand selectivities. In view of this, the regulation of the
LDL receptor (mainly expressed in parenchymal cells) and the mannose
receptor (in nonparenchymal cells) may involve different estrogen
receptors, whether it be homodimeric forms of ER or ER or
heterodimers.48
In conclusion, the increased t-PA clearance after estrogen treatment
via increased mannose receptor expression as shown in this study
provides an explanation for the lowered t-PA levels associated with
high estrogen status. The data presented in this study demonstrate for
the first time that estrogens, besides their well-known effects on
plasma lipid levels via changes in clearance receptors, are also able
to modulate the hemostatic system via changes in clearance rate.
Further research, including identification of the estrogen receptor(s)
involved, is required to precisely analyze the estrogen-regulated
mannose receptor gene expression at the molecular level.
| |
ACKNOWLEDGMENT |
The authors are grateful to Dr Thomas Willnow and Dr Joachim Herz for
providing the adenovirus containing RAP and -GAL cDNA. We thank
Vivian Dahlmans, Marijke Voskuilen, and Richard van Veghel for
excellent technical assistance.
| |
FOOTNOTES |
Submitted October 20, 1998; accepted April 15, 1999.
Supported by grants from the Netherlands Heart Foundation (92.324) and
the Netherlands Organization for Scientific Research, Council for
Medical Research, Medical Sciences (903.39-117).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Teake Kooistra, PhD, Gaubius Laboratory,
TNO Prevention and Health, PO Box 2215, 2301 CE Leiden, The
Netherlands.
| |
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