Vol. 275, Issue 3, H1011-H1015, September 1998
Estrogens modulate bovine vascular endothelial cell
permeability and HSP 25 expression concomitantly
F.
Delarue1,
S.
Daunes1,
R.
Elhage1,
A.
Garcia2,
F.
Bayard1, and
J.-C.
Faye1
1 Institut National de la
Santé et de la Recherche Médicale Unité 397, Institut
L. Bugnard, Centre Hospitalier Universitaire (CHU) Rangueil, 31403 Toulouse Cedex 4; and 2 Institut
National de la Santé et de la Recherche Médicale
Unité 326, CHU Purpan, 31059 Toulouse Cedex, France
 |
ABSTRACT |
The atheroprotective properties of estrogens
are supported by clinical data from postmenopausal women who use
estrogen replacement therapy. However, the mechanisms mediating
activity remain unknown, and it has been suggested that estrogens may
help to modulate endothelial permeability to atherogenic lipoproteins.
In these studies we used bovine vascular endothelial cells as an in
vitro model to show that estrogens were able to regulate low-density lipoprotein transport and permeability of the endothelial monolayer. Macromolecular transport was observed to be a second-order polynomial function of estrogen concentration. Moreover, this regulation was
correlated with expression of heat shock protein (HSP) 25, which is
known to influence fluid phase pinocytosis and cytoskeleton remodeling,
thus suggesting a role for HSP 25 in the estrogenic control of
transcellular permeability of the endothelium monolayer.
atherosclerosis; endothelial permeability; bidimensional
electrophoresis
| |
INTRODUCTION |
THE ATHEROPROTECTIVE EFFECT of estrogens
is supported by abundant epidemiologic data, which has prompted
recommendations for their widespread use in postmenopausal replacement
therapy (19). However, the mechanisms mediating such protection remain
obscure. This protection has traditionally been thought to be caused by potentially favorable changes in blood lipids and lipoproteins (19),
although a number of animal studies strongly suggest a direct effect on
the vascular system (1, 11, 12, 30). It has been suggested in this
context that alterations in endothelial permeability may contribute to
lesion development as a complement to retention of the infiltrated
material (see Refs. 21 and 32 for review) and that suppression of the
accumulation and/or degradation of atherogenic lipoproteins at
this level may be important in the estrogenic effect (12, 30). We have
recently reported that bovine vascular endothelial cells are estrogen
targets that express aromatase, 17
-estradiol hydroxysteroid
dehydrogenase, and 17-ketoreductase enzyme activities and estrogen
receptors and in which ethynylestradiol
(EE2) inhibits superoxide anion production (2, 3). We decided to use this in vitro experimental model
to study the effect of estrogens on cell permeability and the
extracellular matrix retention of low-density lipoproteins (LDL).
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MATERIALS AND METHODS |
Chemicals and supplies.
All tissue culture reagents were supplied by GIBCO. Antibodies directed
against HSP 27, which cross-react with bovine HSP 25, were obtained
from Stressgene, and anti-nitric oxide synthase (NOS) was from
Transduction Laboratories (Lexington, KY).
LDL were isolated from human plasma by sequential isopycnic
ultracentrifugation and were radioiodinated using iodine monochloride as previously described (7).
125I-labeled LDL specific
radioactivity was ~5,000
counts · min
1 · ng1,
which was adapted to the experimental conditions by diluting with
unlabeled LDL.
Lipoprotein lipase (LPL) was purified from fresh, unpasteurized cow's
milk using heparin-agarose affinity chromatography (28). At the time of
utilization, the LPL preparation had a specific activity of 11 U/mg
protein.
Cell culture and preparation of monolayers.
Stock cultures of bovine aortic endothelial cells (BAEC) were obtained
from castrated males and were maintained as previously described (3).
For preparation of monolayers, the cells were grown on 10-mm
polycarbonate filters (pore diameter 3 or 0.02 µm depending on the
experiment; Nucleopore) coated with laminin (100 µl of 20 µg
laminin/ml DMEM; Sigma) as suggested by the manufacturer. Each
laminin-coated filter was seeded with 5 × 105 cells in 500 µl of phenol
red-free DMEM (GIBCO) containing 10% charcoal-stripped bovine calf
serum, antibiotics (gentamicin and Fungizone), glutamine (1%), and 5 ng/ml fibroblast growth factor (FGF)-2. Filters were
disposed in 24 multiwell plates (Nunc) containing 500 µl of culture
medium for 5 days, and the medium of the well was replaced on
day 3 with the same medium without
FGF-2. Cells were treated with hormones or antihormones from
day 2 onward unless otherwise
specified.
Transport studies of 125I-labeled LDL.
Transport studies of 125I-labeled
LDL were carried out as described by Saxena et al. (26). Filters of
3-µm pore diameter were used in these experiments. After the cells
reached confluency they were treated with
EE2
(109 M) for 48 h; controls
were obtained in the absence of
EE2. Culture media from both
chambers were aspirated, and the cells were carefully washed twice with
DMEM containing 3% BSA (DMEM-BSA). Purified LPL (20 µg/ml) was added
to the medium in the upper chamber, and the cells were incubated for 45 min at 37°C and then washed with DMEM-BSA to remove unbound LPL.
After addition of 125I-labeled LDL
to the upper chamber and further incubation at 37°C for 1 h, we
determined the radioactivity in both chambers. The cells were washed
twice with cold DMEM-BSA, and DMEM containing 50 U/ml of heparin (grade
I-A, Sigma) was added to the well for 10 min at 4°C to determine
the 125I-labeled LDL released by
the subendothelial cell matrix.
Transendothelial exchange of fluorescein-labeled dextran.
An endothelial monolayer selectively restricts molecules according to
their size, consistent with a two-pore model with radii of 65 and 304 Å (27). Polycarbonate membranes of 0.02-µm pore size coated
with laminin were used in subsequent studies because of the better
stability of the cell monolayer on this substrate. Fluorescein-labeled
dextran (FD40, mol. wt. 40,000; Sigma) belongs to the same size
category as LDL (27) and was used for examination of the diffusion
kinetics at a concentration of
105 M in DMEM. This tracer
could be directly added to the filter well without changing the culture
medium, thus diminishing the risk of artifactual disruption of the
endothelial barrier. It also permitted elimination of charge
selectivity, uptake, or metabolism by endothelial cells (33). The
diffusion of these molecules across the endothelial monolayer from the
luminal to the abluminal side for 1 h at 37°C was measured in a
fluorimeter (Perkin Elmer) using 495 nm as the excitation wavelength
and 530 nm as the emission wavelength.
The changes in endothelial permeability were studied after cell
incubation with 106 M
l-isoproterenol (Sigma) or with
107 M phorbol 12-myristate
13-acetate (PMA; Sigma) for 15 min before permeability measurement.
EE2 effects were assessed by
kinetic studies using increasing concentrations in the culture medium (1010-108
M added from 1,000× ethanolic solution) for increasing periods of
time (12-72 h). To verify estrogen receptor involvement in the
EE2 effect, experiments were
performed with various compounds used alone or in combination:
17-estradiol (109 M),
17
-estradiol (108 M),
progesterone (108 M),
testosterone (108 M),
cortisol (108 M), and
antiestrogens at concentrations able to displace the estrogen
completely from its receptor, tamoxifen (5 × 107 M) or RU-54876 (5 × 107 M).
Western blot analysis and two-dimensional gel electrophoresis.
After estrogen treatment
(109 M for 2 days), cells
were scraped in PBS with a rubber policeman, pelleted at 1,700 g and lysed at 100°C (10 min) in
2% SDS buffer. Five-microgram proteins were separated on 12.5%
SDS-PAGE at 120 V and then electroblotted onto a nitrocellulose (0.45 µm) membrane with a Bio-Rad transblot system for 75 min at 100 V. After transfer, the proteins were reversibly stained with Ponceau red.
The membrane was then blocked with 3% fat-free milk in Tris-borate
buffer (TBST; pH 8) for 1 h and incubated with anti-HSP 27 (1:2,000) antibody, washed three times with TBST for 10 min and
incubated with secondary antibody coupled to horseradish peroxidase,
washed four more times, and then visualized by detection of
chemiluminescence with an Amersham kit. The same nitrocellulose filter
was used for NOS III immunodetection.
In another series of experiments cells were harvested in calcium- and
magnesium-free phosphate buffer saline and then centrifuged and
resuspended in Tris · HCl buffer (50 mM, pH 7.4)
containing 2.5 mM EDTA, 70 mM sucrose, 210 mM
D-mannitol, 1 µg/ml leupeptin, 1 µg/ml epibestatin, 0.5 µg/ml trypsin inhibitor, 0.1 mM
phenylmethylsulfonyl fluoride, and 0.5 µg/ml aprotinin. Cells were
sonicated at 4°C in this buffer and centrifuged at 105,000 g for 1 h. The protein supernatant
concentration was measured by bicinchoninic acid assay reagent.
Analytical two-dimensional (2-D) gel electrophoresis was performed as
described by O'Farrell (22) with slight modifications. Briefly, the
isoelectrofocusing (IEF) gels were composed of 9.5 M urea, 4.2%
ampholytes (0.6% pH 3-10, 0.6% pH 2-11, 1.2% pH 4-6, 1.8% pH 5-8), 4% acrylamide, 0.24% bisacrylamide, 5%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.02% ammonium persulfate, and 0.0013%
N,N,N',N'-tetramethylethylenediamine. The anode solution was 0.065% phosphoric acid, and the cathode solution was 0.1 M sodium hydroxide. Proteins were loaded in 2-D buffer
[2% ampholytes 5-8, 2% CHAPS, 9.5 M urea, 0.1 M
dithiothreitol (DTT)], and 40 µg of each sample were subjected
to IEF for 18 h at 1,000 V. After completion of the IEF, gels
were equilibrated in a solution of 0.05 M Tris · HCl
pH 6.8, 2% SDS, 8 mM bromophenol blue, 0.77% DTT, and 0.37%
iodoacetamide for 4 min. The gels were then run on 12.5%
SDS-PAGE. After a 5-h run the gels were fixed in 40% methanol, 10%
acetic acid, and 5% Formalin for at least 2 h under agitation. They
were stained with silver nitrate according to the method previously
described (25) and then scanned and quantitated with the Bioimage
apparatus (2-D Analyzer). Cartesian coordinates [relative
molecular weight
(Mr),
isoelectric point (pI)] were compared with a 2-D database
(20).
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RESULTS |
Effect of EE2 on transport of
125I-labeled LDL and retention by
endothelial cell monolayer.
In agreement with the observations of Saxena et al. (26), LDL transport
across the endothelial cell monolayers appeared as a nonsaturable
process in the range of 0.1-80 µg
125I-labeled LDL/ml (data not
shown). Figure
1A shows
that EE2
(109 M for 48 h)
significantly decreased LDL transport [2.31 ± 0.02 vs. 2.00 ± 0.11 ng/h and 415.3 ± 21.1 vs. 359.0 ± 25.8 ng/h (mean ± SD), n = 10 experiments; P < 0.01] when
0.5 or 72 µg/ml 125I-labeled
LDL, respectively, was added to the filters. In contrast (Fig. 1B), LDL retention by the
subendothelial cell matrix, in the absence or presence of
EE2, was not statistically
significant (0.12 ± 0.04 vs. 0.11 ± 0.02 and 9.1 ± 0.2 vs.
9.5 ± 0.2 ng/h when 0.5 or 72 µg/ml
125I-labeled LDL, respectively,
was added, n = 10 experiments, P > 0.1).
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Fig. 1.
Low-density lipoprotein (LDL) transport across endothelial cell
monolayer (A) and retention
(B) by subendothelial cell matrix.
Transport of LDL across confluent monolayers of bovine endothelial
cells treated with ethynylestradiol
(EE2;
109 M for 48 h) or not (C)
was studied using 0.5 and 72 µg of
125I-labeled LDL at 37°C for
45 min. Amounts transported (A) and
retained (B) by monolayers are
indicated. Results are means ± SD of 10 different measurements.
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As previously described (33), BAEC showed an increasing tightness of
the monolayer when stimulated with 1 µM l-isoproterenol. In contrast, endothelial permeability was increased (+67%; mean of 3 experiments) when BAEC were treated with 0.1 µM PMA (not shown). As
shown in Fig. 2, a biphasic effect of
estrogens on permeability was observed after 2-day treatment and a
statistically significant second-order polynomial relationship between
increasing concentrations of EE2
treatment for 72 h and FD40 transfer could be characterized
(r = 0.866, P < 0.005). The maximal decrease, obtained at 109 M, reached
the values obtained using 1 µM l-isoproterenol and was not
additive with the effect of the catecholamine. The increased permeability induced by PMA treatment was not significantly affected by
EE2. The decreased permeability
induced by EE2
(109 M) was time dependent
and only detectable between 24 and 48 h of treatment (Fig.
3); 17-estradiol was also active,
whereas 17-estradiol as well as other nonestrogen steroid hormones,
progesterone, testosterone and cortisol (not shown), were inactive.
Antiestrogens of triphenylethylene (tamoxifen) or steroid (RU-54876)
structure did not display any effect on their own but were both
antagonists of EE2 (Fig. 3).
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Fig. 2.
Dose-dependent kinetics of 40-kDa fluorescein-labeled dextran (FD40)
transport across endothelial cell monolayers under
EE2 with ( ) or without ( )
l-isoproterenol
(106 M) treatment.
Endothelial cells were treated or not (C) with increasing
concentrations of EE2 for 48 h.
FD40 (106 M) was added to
luminal compartment. After 1 h at 37°C, amount of tracer in
abluminal compartment was determined in arbitrary units (a.u.). Data
presented are duplicate measurements from same experiment and are
representative of 3 independent experiments.
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Fig. 3.
Time-dependent kinetics of FD40 transport across endothelial cell
monolayers under EE2 treatment.
Endothelial cells were treated with
109 M
EE2 for various periods of time or
for 48 h with different molecules, and then FD40 was added in luminal
compartment. After 1 h at 37°C, amount of tracer in abluminal
compartment was determined as in Fig. 2. Data presented are duplicate
measurements in same experiment and are representative of 3 independent
experiments. , EE2
(109 M);  ,
17-estradiol (109 M);
, 17-estradiol (108
M);  , EE2
(109 M) + RU-54876 (5 × 107 M);  ,
EE2
(109 M) + tamoxifen (5 × 107 M).
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Effect of estrogens on regulation of HSP 25 expression.
Because the small heat shock protein HSP 25/27 has been shown to
control pinocytosis (6, 17, 18) and to be expressed under estrogen
control in vascular endothelial cells (24), we thought it would be of
interest to study the expression of this protein under the conditions
of our studies. Western blot analysis showed (Fig.
4) that, at concentrations up to
109 M,
EE2 treatment for 48 h decreased
HSP 25 expression, which was restored in a biphasic fashion at 5 109 M. Higher
EE2 concentrations increased
protein synthesis above the basal level. NOS III was used as the
internal marker (Fig. 4) because we previously showed that
EE2 had no effect on cell concentrations of this protein (2). It was clearly apparent that a
109 M
EE2 treatment induced a fourfold
decrease in the relative expression of HSP 25. Antiestrogens prevented
this decrease (data not shown).
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Fig. 4.
Regulation of HSP 25 production by increasing
EE2 concentrations.
Top: Western blot analyses of heat
shock protein (HSP) 25 and nitric oxide synthase (NOS) III were carried
out on the same nitrocellulose filter as described in
MATERIALS AND METHODS.
Bottom: relative quantitation of HSP
25/NOS III.
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The effects of EE2
(109 M for 2 days) on the
bidimensional electrophoresis pattern of BAEC proteins were also
analyzed. Computer analyses of six independent experiments using the
Bioimage 2-D Analyzer program revealed a 5-fold variation for 12 spots
and >10-fold variation for another spot under
EE2 treatment, but the greatest
difference between the electrophoretic patterns of treated and
nontreated cells resided in the disappearance of a polypeptide of
Mr 25,000, pI 5.9 under estrogenic treatment (identified by arrow in Fig.
5). These biophysical properties confirmed
that this spot corresponded to HSP 25 (20), showing that, even if HSP
25 was not the sole protein modulated by
EE2, it constituted the major
change noted with 2-D electrophoresis. The greater variation evidenced
with 2-D electrophoresis, compared with the Western blotting data,
probably resulted from the different cell fraction analyzed
and/or the use of different visualization processes. These
results in relation with the previously described function of this
protein on the regulation of fluid phase pinocytosis (18) suggest that
it could be involved in the modulation of LDL transport.
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Fig. 5.
Two-dimensional gel electrophoresis analyses of extracts from
endothelial cells treated or not (control) with
EE2. Procedure was carried out as
described in MATERIALS AND METHODS.
Isoelectric point (pI, abscissa) and molecular mass (MM, ordinate) were
calibrated with an external marker calibration kit (BioRad). Arrow
indicates spot that disappeared after
EE2
(109 M) treatment.
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DISCUSSION |
In agreement with previous in vitro (26) as well as in vivo (31)
studies, our data confirm that LDL crosses the endothelium in an LDL
receptor-independent fashion. They also demonstrate that
EE2 can decrease the permeability
of cultured BAEC monolayers to LDL but has no effect on LDL retention
by the subendothelial cell matrix. Endothelial cell monolayers have
provided a useful model for examining the components of the response to
estrogens. Under these conditions,
109 M
EE2 was unable to reverse the
permeability-increasing effect of PMA, which induces cell contraction,
thereby producing intercellular gaps (23). It did, however, develop an
activity comparable to but not additive with isoproterenol, which
decreases paracellular and possibly also transcellular pathways of
endothelial permeability involving actin filaments (21, 23, 33). Among
the number of actin-binding proteins concerned with the regulation of
actin polymerization and organization, HSP 25/27, which belongs to the family of small heat shock proteins, has been shown to control pinocytosis (17, 18) and to be expressed in vascular endothelial cells
under estrogen control (24). We have now demonstrated that the HSP 25 protein content of bovine endothelial cells varies in parallel with the
transendothelial permeability in a second-order polynomial relationship
and that this variation also appears to be regulated via an estrogen
receptor, although we cannot yet conclude from these experiments
whether the variation in cell content resulted from decreased
production or increased catabolism of the protein. Estradiol treatment
of BAEC results in an increase in HSP 25, with peak expression at 100 nM (24). Our data agree with such an increase at high estradiol
concentration. However, they also show the decrease induced at low and
more physiological estradiol concentrations concomitantly with the
decrease in endothelial permeability. The precise significance of these
biphasic phenomena must be determined. They may represent differential
activities of a single receptor or combinatory effects of the two
estrogen receptors characterized in this population of cells (3, 9).
The accumulation of atherogenic lipoproteins in the arterial wall
intima constitutes a fundamental event in atherogenesis (21, 32), and
it has been suggested that estrogens could be involved in this process
(30). However, the observed effects of estrogens on LDL transport were
not dramatic, thus questioning the functional significance of this
pathway, which has also been questioned in in vivo experiments (10). In
fact, distribution of the early lesions of atherosclerosis is
nonrandom, occurring at arterial bifurcations and curvatures, where the
disturbed flow patterns lead to the development of potential
proinflammatory and proatherogenic activities (8) with transient leaky
junctions surrounding mitotic or dying endothelial cells (5). In these areas, the passage of lipoproteins across the endothelial monolayer would probably reflect accelerated cell turnover (4) rather than
endothelial permeability (15, 29) and estrogens could develop more
significant effects by interfering with apoptosis (14) and increasing
proliferation rate (16). The effects of estrogens evidenced in our
studies could have different pathophysiological implications such as,
for example, in the production of reactive oxygen species (2, 13)
and/or production of oxidized LDL.
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ACKNOWLEDGEMENTS |
This work was supported by Ligue Nationale contre le Cancer,
Institut National de la Santé et de la Recherche Médicale, and la région Midi-Pyrénées. F. Delarue is a
fellowship recipient from Ligue régionale du Tarn et Garonne.
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FOOTNOTES |
Address for reprint requests: J.-C. Faye, Institut Louis Bugnard,
INSERM U397, CHU Rangueil 31403 Toulouse Cedex 4, France.
Received 19 December 1997; accepted in final form 1 June 1998.
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REFERENCES |
1.
Adams, M. R.,
J. R. Kaplan,
S. B. Manuck,
D. R. Koritnik,
J. S. Parks,
M. S. Wolfe,
and
T. B. Clarkson.
Inhibition of coronary artery atherosclerosis by 17-beta estradiol in ovariectomized monkeys. Lack of an effect of added progesterone.
Arteriosclerosis
10:
1051-1057,
1990[Abstract/Free Full Text].
2.
Arnal, J. F.,
S. Clamens,
C. Pechet,
A. Negre-Salvayre,
C. Allera,
J. P. Girolami,
R. Salvayre,
and
F. Bayard.
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.
Bayard, F.,
S. Clamens,
G. Delsol,
N. Blaes,
A. Maret,
and
J. C. Faye.
Non-Reproductive Actions of Sex Steroids. Chichester, UK: Wiley, 1995, p. 122-138. (Ciba Fndn. Symp. 191)
4.
Chang, E.,
and
C. B. Harley.
Telomere length and replicative aging in human vascular tissues.
Proc. Natl. Acad. Sci. USA
92:
11190-11194,
1995[Abstract/Free Full Text].
5.
Chen, Y. L.,
K. M. Jan,
H. S. Lin,
and
S. Chien.
Relationship between endothelial cell turnover and permeability to horseradish peroxidase.
Atherosclerosis
133:
7-14,
1997[Medline].
6.
Ciocca, D. R.,
S. Oesterreich,
G. Chamness,
W. L. McGuire,
and
S. A. Fuqua.
Biological and clinical implications of heat shock protein 27,000 (Hsp27): a review.
J. Natl. Cancer Inst.
85:
1558-1570,
1993[Abstract/Free Full Text].
7.
Garcia, A.,
R. Barbaras,
X. Collet,
A. Bogyo,
H. Chap,
and
B. Perret.
High-density lipoprotein 3 receptor-dependent endocytosis pathway in a human hepatoma cell line (HepG2).
Biochemistry
35:
13064-13071,
1996[Medline].
8.
Gimbrone, M. A., Jr.,
T. Nagel,
and
J. N. Topper.
Biomechanical activation: an emerging paradigm in endothelial adhesion biology.
J. Clin. Invest.
99:
1809-1813,
1997[Medline].
9.
Gustafsson, J. A.
Estrogen receptor : getting in on the action.
Nat. Med.
3:
493-494,
1997[Medline].
10.
Haarbo, J.,
L. B. Nielsen,
S. Stender,
and
C. Christiansen.
Aortic permeability to LDL during estrogen therapy. A study in normocholesterolemic rabbits.
Arterioscler. Thromb.
14:
243-247,
1994[Abstract/Free Full Text].
11.
Hayashi, T.,
J. M. Fukuto,
L. Ignarro,
G. Chaudhuri,
and
T. Hayashi.
Basal release of nitric oxide from aortic rings is greater in female rabbits than in male rabbits: implications for atherosclerosis.
Proc. Natl. Acad. Sci. USA
89:
11259-11263,
1992[Abstract/Free Full Text].
12.
Hough, J. L.,
and
D. B. Zilversmit.
Effect of 17 beta estradiol on aortic cholesterol content and metabolism in cholesterol-fed rabbits.
Arteriosclerosis
6:
57-62,
1986[Abstract/Free Full Text].
13.
Huot, J.,
F. Houle,
F. Marceau,
and
J. Landry.
Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells.
Circ. Res.
80:
383-392,
1997[Abstract/Free Full Text].
14.
Kaiser, D.,
M. A. Freyberg,
and
P. Friedl.
Lack of hemodynamic forces triggers apoptosis in vascular endothelial cells.
Biochem. Biophys. Res. Commun.
231:
586-590,
1997[Medline].
15.
Kao, C. H.,
J. K. Chen,
J. S. Kuo,
and
V. C. Yang.
Visualization of the transport pathways of low density lipoproteins across the endothelial cells in the branched regions of rat arteries.
Atherosclerosis
116:
27-41,
1995[Medline].
16.
Krasinski, K.,
I. Spyridopoulos,
T. Asahara,
R. van der Zee,
J. Isner,
and
D. W. Losordo.
Estradiol accelerates functional endothelial recovery after arterial injury.
Circulation
95:
1768-1772,
1997[Abstract/Free Full Text].
17.
Lavoie, J. N.,
G. Gingras-Breton,
R. M. Tanguay,
and
J. Landry.
Induction of Chinese hamster HSP27 gene expression in mouse cells confers resistance to heat shock. HSP27 stabilization of the microfilament organization.
J. Biol. Chem.
268:
3420-3429,
1993[Abstract/Free Full Text].
18.
Lavoie, J. N.,
E. Hickey,
L. A. Weber,
and
J. Landry.
Modulation of actin microfilament dynamics and fluid phase pinocytosis by phosphorylation of heat shock protein 27.
J. Biol. Chem.
268:
24210-24214,
1993[Abstract/Free Full Text].
19.
Lobo, R. A.,
and
L. Speroff.
International consensus conference on postmenopausal hormone therapy and the cardiovascular system.
Fertil. Steril.
61:
592-595,
1994[Medline].
20.
Muller, E. C.,
P. Thiede,
U. Zimmy-Arndt,
C. Scheler,
J. Prehm,
U. Mueller-Werdan,
B. Wittmann-Liebold,
A. Otto,
and
P. Jungblut.
High-performance human myocardial two-dimensional electrophoresis database.
Electrophoresis
17:
1700-1712,
1996[Medline].
21.
Nielsen, L. B.
Transfer of low density lipoprotein into the arterial wall and risk of atherosclerosis.
Atherosclerosis
123:
1-15,
1996[Medline].
22.
O'Farrell, P. H.
High resolution two-dimensional electrophoresis of proteins.
J. Biol. Chem.
250:
4007-4021,
1975[Abstract/Free Full Text].
23.
Oliver, J. A.
Adenylate cyclase and protein kinase C mediate opposite actions on endothelial junctions.
J. Cell. Physiol.
145:
536-542,
1990[Medline].
24.
Piotrowicz, R. S.,
L. A. Weber,
E. Hickey,
and
E. G. Levin.
Accelerated growth and senescence of arterial endothelial cells expressing the small molecular weight heat-shock protein HSP 27.
FASEB J.
9:
1079-1084,
1995[Abstract].
25.
Rabilloud, T.,
G. Carpentier,
and
P. Tarroux.
Improvement and simplification of low-background silver staining of proteins by using sodium dithionite.
Electrophoresis
9:
288-291,
1988[Medline].
26.
Saxena, U.,
M. G. Klein,
T. M. Vanni,
and
I. J. Goldberg.
Lipoprotein lipase increases low density lipoprotein retention by subendothelial cell matrix.
J. Clin. Invest.
89:
373-380,
1992.
27.
Siflinger-Birboim, A.,
P. J. Del Vecchio,
J. A. Cooper,
F. A. Blumenstock,
J. M. Shepard,
and
A. B. Malik.
Molecular sieving characteristics of the cultured endothelial monolayer.
J. Cell. Physiol.
132:
111-117,
1987[Medline].
28.
Simard, G.,
B. Perret,
S. Durand,
X. Collet,
H. Chap,
and
L. Douste-Blazy.
Phosphatidylcholine and triacylglycerol hydrolysis in HDL as induced by hepatic lipase: modulation of the phospholipase activity by changes in the particle surface or in the lipid core.
Biochim. Biophys. Acta
1001:
225-233,
1989[Medline].
29.
Van Hinsbergh, V. W. M.
Endothelial permeability for macromolecules: mechanistic aspects of pathophysiological modulation.
Arterioscler. Thromb. Vasc. Biol.
17:
1018-1023,
1997[Free Full Text].
30.
Wagner, J. D.,
T. B. Clarkson,
R. W. St. Clair,
D. C. Schwenke,
C. A. Shively,
and
M. R. Adams.
Estrogen and progesterone replacement therapy reduces low density lipoprotein accumulation in the coronary arteries of surgically postmenopausal cynomolgus monkeys.
J. Clin. Invest.
88:
1995-2002,
1991.
31.
Wicklund, O.,
T. E. Carew,
and
D. Steinberg.
Role of the low density lipoprotein receptor in penetration of low density lipoprotein into rabbit aortic wall.
Arteriosclerosis
5:
135-141,
1985[Abstract/Free Full Text].
32.
Williams, K. J.,
and
I. Tabas.
The response-to-retention hypothesis of early atherogenesis.
Arterioscler. Thromb. Vasc. Biol.
15:
551-561,
1995[Free Full Text].
33.
Zink, S.,
P. Rösen,
and
H. Lemoine.
Micro- and macrovascular endothelial cells in -adrenergic regulation of transendothelial permeability.
Am. J. Physiol.
269 (Cell Physiol. 38):
C1209-C1218,
1995[Abstract/Free Full Text].
Am J Physiol Heart Circ Physiol 275(3):H1011-H1015
0002-9513/98 $5.00
Copyright © 1998 the American Physiological Society
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Abstract
Introduction
