(Circulation. 1995;91:755-763.)
© 1995 American Heart Association, Inc.
Articles |
Estrogen Promotes Angiogenic Activity in Human Umbilical Vein Endothelial Cells In Vitro and in a Murine Model
From the Laboratory of Developmental Biology, National Institute of Dental Research (D.E.M., K.A.M., D.S.G., S.M., M.C.C., H.K.K., H.W.S.), and the Developmental Endocrinology Branch, National Institute of Child Health and Human Development (D.B.), National Institutes of Health, Bethesda, Md; the Department of Internal Medicine (M.C.C.), Hospital Clinic i Provincial, Barcelona, Spain; and the Department of Pediatrics (H.W.S.), Northwestern Medical School.
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Abstract |
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Background Angiogenesis is a critical event in wound healing, tumor growth, and the inflammatory vasculitides. Since women have a higher incidence of many vasculitic diseases, we examined the effects of female sex steroids, particularly estradiol, on human umbilical vein endothelial cell (HUVEC) behavior in vitro and on angiogenesis in vivo.
Methods and Results HUVECs were grown in estrogen-free medium before each assay. Exogenous 17ß-estradiol (1 to 5 nmol/L) increased cell attachment to laminin, types I and IV collagen, and fibronectin, as well as to tissue culture plastic. After a confluent monolayer of cells was "wounded" by scraping, estradiol-treated (10-8 mol/L) cells migrated into the wound three times faster than untreated cells. Cell proliferation on plastic and on laminin increased threefold to fivefold, respectively, in the presence of estradiol. Estradiol also enhanced the ability of HUVECs to organize into tubular networks when plated on a reconstituted basement membrane, Matrigel. Estradiol effects on both the "wounding" assay and tube formation were blocked by the specific estrogen receptor antagonist ICI 182,780. Ovariectomy markedly decreased in vivo vascularization of Matrigel plugs coinjected with basic fibroblast growth factor in mice. With estrogen replacement, angiogenesis was increased to the levels observed in nonovariectomized mice.
Conclusions These studies demonstrate that, in vitro and in vivo, estradiol enhances endothelial cell activities important in neovascularization and suggest a promoting influence of estrogens on angiogenesis.
Key Words: angiogenesis • endothelium • estrogen • cells
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Introduction |
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Angiogenic activity is generally low in the adult organism, except for the normal cyclical changes that occur in the female reproductive tract.1 Only during injury and certain pathological conditions do other tissues exhibit angiogenesis. For example, in arthritis,2 capillaries invade and destroy joint cartilage. In diabetic retinopathy, vessels invading the corpus vitreum lead to blindness.3 Neovascularization also occurs during both wound healing4 5 and solid tumor growth.6 Generally, initiation of blood vessel formation involves several steps, beginning with enzymatic degradation of the associated basement membrane. Vascular endothelial cells then migrate into the stromal space, proliferate, and align. The cells form tubular structures, undergo significant remodeling, and finally reestablish a new basement membrane.7 8
Several manifestations of vascular disease suggest that endothelial cell activity, especially angiogenesis, may be regulated by sex hormones. For example, systemic lupus erythematosus (SLE)9 and Takayasu's arteritis10 occur most often in women of childbearing age. Both of these diseases are associated with endothelial cell proliferation.11 SLE may be exacerbated by either pregnancy or the use of oral contraceptives. Also, experimental SLE in NZB/W mice is more severe in females than in males,2 11 12 and the outcome can be affected by castration and/or hormonal replacement. The role of the endothelium in mediating cellular immune responses suggests that the effect of sex steroids, particularly estrogens, may be mediated at least in part through effects on endothelial cells.13
A role for gonadal steroids in modulating endothelial cell behavior is supported by studies of blood vessel formation in the primate endometrium.14 It is likely that neovascularization in reproductive tissues is under the control of steroid hormones, particularly 17ß-estradiol in women.15 16 In the present article, we report studies of the effect of estradiol on behavior of nonendometrial endothelial cells both in vivo and in vitro. Our results suggest that estrogens enhance angiogenesis by promoting cell attachment, migration, proliferation, and differentiation.
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Methods |
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Materials
Unless indicated otherwise, all materials were reagent grade and purchased from commercial sources.
Endothelial Cell Culture
Human umbilical vein endothelial
cells (HUVECs) were obtained by
established methods from freshly delivered umbilical cords obtained
after cesarean births.17 Initially, cells were grown in a
standard endothelial cell culture medium consisting of Medium 199
(M-199) (Gibco) supplemented with 20% bovine calf serum (BCS) (Hyclone
Laboratories), 100 U/mL penicillin/streptomycin, 50 µg/mL gentamycin,
2.5 µg/mL amphotericin B, 2 mmol/L glutamine, 5 U/mL sodium heparin,
and 200 µg/mL endothelial cell growth supplement (ECGS)
(Collaborative Research). The latter is an extract of bovine brain,
rich in acidic fibroblast growth factor,18 that is
essential for endothelial cell growth in vitro.19
Immunostaining for factor VIII–related antigen (von Willebrand factor,
vWF)20 confirmed that the cells were endothelial. Cells
used for experiments were from passages 4 through 8 and were switched
to a hormone-free growth medium 48 hours before each
assay.13 Because of the estrogen-like effects of
phenol,21 the hormone-free medium was prepared with
phenol-free improved minimum essential medium (IMEM) (Biofluids Inc)
and 20% BCS incubated with dextran-treated activated
charcoal.22 This procedure removes several biologically
active molecules from the serum, including endogenous estrogens. The
medium was then supplemented with the same reagents as the standard
endothelial cell culture medium.
Reagents Used to Study Hormonal Effects
17ß-Estradiol,
progesterone, and testosterone were obtained
from Sigma. Stock solutions in absolute ethanol were stored at
-20°C. Appropriate dilutions in IMEM were then made and applied
directly to cultures. Concentrations tested usually ranged from 0.1 to
5 ng/mL (3x10-10 to 1x10-8
mol/L); this
range of concentrations is similar to those found in most men and
nonpregnant women.23 Control cultures received the ethanol
vehicle (0.1%) alone. The specific estrogen receptor antagonist ICI
182,780 was provided by Zeneca Pharmaceuticals. A stock solution in
100% ethanol was diluted in phenol-free IMEM before addition to
cultures at the same time as estradiol; no effect was seen when vehicle
was added alone (<0.01% final concentration).
Attachment Assay
A modification of the Klebe
assay24 was used.
HUVECs grown in hormone-free medium for 48 hours were cultured for 3
hours with various amounts of 17ß-estradiol, progesterone, or
testosterone. The cells were rinsed with Dulbecco's phosphate-buffered
saline (DPBS) (Biofluids), detached from the culture dish with EDTA in
PBS (Versene, GIBCO), pelleted, and resuspended in serum-free IMEM.
Various test proteins, including laminin (10 µg/well) prepared from
the murine EHS tumor25 ; fibronectin (12.5 µg/well) (a
gift of S. Akiyama, NIDR); collagen I (5 µg/well) (Collaborative
Research); and collagen IV (10 µg/well) prepared from EHS tumors in
lathritic mice as described previously,26 were coated on
24-well plates (Costar). The plates were subsequently incubated for 30
minutes with 3% bovine serum albumin (Boehringer Mannheim) to block
nonspecific binding. The liquid was aspirated, and 8x104
cells/well were plated. Estradiol was added directly to the wells. At
various times after incubation at 37°C, the medium was aspirated, and
the adherent cells were rinsed twice with DPBS before being fixed and
stained with Diff-Quik (Baxter Healthcare Corp). The average area per
well of adherent cells was measured in triplicate wells at x25
magnification with a computerized digital image analyzer (Optomax V HR)
connected to an Olympus microscope.27 Each data point was
tested in triplicate, and the assays were performed three times.
Results were expressed as the mean cell area per field ±SEM (relative
units).
Wounding Migration Assay
HUVECs cultured for 48 hours in
hormone-free medium were plated
at about 90% confluence in six-well culture plates (Nunc, Intermed).
After 12 hours, the endothelial monolayers were wounded28
by two perpendicular strokes across the diameter of the well with a
2.5-mm-wide cell scraper. The media and dislodged cells were aspirated,
and the plates were rinsed with DPBS. Fresh hormone-free medium was
added to the plates along with 17ß-estradiol at various
concentrations. Thymidine (10 mmol/L) was also added to inhibit cell
proliferation to observe the effects of migration rather than cell
proliferation on wound closure. The cells were incubated at 37°C. At
various times after wounding, plates were fixed and stained with
Diff-Quik. The width of the wound was visualized with a Nikon
Optiphot-2 microscope at x10 magnification and was quantified with the
NIH IMAGE 1.47 processing and analysis program for
the Macintosh. A real-time image for analysis was acquired directly
from the microscope onto a QuikCapture board (Data Translation, Inc). A
total of 20 random measurements were analyzed for each of four wounds
at each time point. Results were expressed as mean percent closure of
the wounds over time ±SEM. At least three experiments were performed.
Serial photographs of plates were taken over a 24-hour time course.
Boyden Chamber Migration Assay
PVPF 13-mm filters with
13-µm pores (Nucleopore) were coated
with 5 µg type IV collagen on their top surface and allowed to dry
overnight. After 24 hours, HUVECs cultured in hormone-free medium were
preincubated with estradiol for 3 hours, then were lifted with Versene
and resuspended in IMEM/0.1% BSA. The lower segment of the chamber was
filled with 250 µL IMEM supplemented with 100 µg ECGS
(Collaborative Research) to serve as the chemoattractant. Cells
(2x105) in IMEM/0.1% BSA were added to the upper
chambers. Estradiol (0.1 to 4 ng/mL) was added to both the upper and
lower chambers at the same concentration. The Boyden chambers were
incubated at 37°C for 5 hours, at which time the filters were
removed, rinsed, fixed, and stained with Diff-Quik.29
Cells on the upper side of the filter were swabbed off. The assay was
quantified by counting of the number of cells per microscopic field
that migrated through the pores to the lower surface of each filter.
Each data point was based on triplicate chambers and three microscopic
fields per filter. Filters were counted with a Nikon-Optiphot-2 at x20
magnification. Each assay was performed three times. Results were
expressed as the mean number of cells per field ±SEM.
Proliferation Assay
HUVECs cultured in hormone-free medium
for 48 hours were lifted
with either 0.05% trypsin-EDTA or Versene and resuspended at 5000
cells/mL in hormone-free medium. Cultures (1 mL) were plated on 24-well
culture plates (Nunc) and allowed to attach. Estradiol was then added
to each well at 1.5 ng/mL, and the cells were cultured at 37°C.
Triplicate wells of cells were lifted and counted with a cell counter
(Coulter Electronics) for 5 consecutive days. Proliferation was
analyzed on plastic and on wells coated with laminin (10 µg/well).
Each assay was performed three times. Results were expressed as the
mean number of cells per well ±SEM.
Organization of HUVECs on a Basement Membrane Substratum In
Vitro
Reconstituted basement membrane Matrigel was prepared from the
murine Engelbreth-Holm-Swarm tumor30 and dialyzed against
phenol-free IMEM. Twenty-four–well plates (Costar) were coated with
320 µL of Matrigel (12 to 16 mg/mL) per well, which was allowed to
polymerize for 30 minutes at 37°C. Into each culture well was added
500 µL IMEM. HUVECs suspended in 500 µL of hormone-free culture
medium were then added to each well to bring the final culture to
4x104 cells in 1 mL. Estradiol was added to the wells at
various concentrations from 0.1 to 5 ng/mL, and the plates were
incubated overnight at 37°C. After removal of the medium by
aspiration, the culture was fixed and stained with DiffQuik. The area
of the tube network on the culture surface was quantified at x10
magnification by the Optomax-Olympus method.27 Results
from triplicate wells were expressed as mean vessel area per field
±SEM (relative units) from 20 random fields per well. Each assay was
performed at least four times.
In Vivo Angiogenesis Model
Sexually mature 6- to 8-week-old
C57Bl/6 female mice weighing 20
to 25 g were bilaterally ovariectomized through a 1-cm dorsal incision.
The peritoneal cavity was entered bilaterally with 0.5-cm incisions.
The ovaries were identified, retracted, and excised. After 9 days, the
mice were randomly allocated to receive a 0.5-mg subcutaneous placebo
pellet or a 17ß-estradiol 21-day slow-release pellet (Innovative
Research of America) prepared to generate a serum estradiol
concentration up to 750 pg/mL; this exceeds the usual physiological
concentration in female mice but is less than levels obtained with
high-dose estrogen treatment for, eg, antitumor therapy. The pellets
were inserted interscapularly under anesthesia. Mock-ovariectomized
mice treated with placebo pellets served as controls.
After recovery, the mice were evaluated for ability to vascularize a Matrigel plug. On day 10 after ovariectomy, they received 0.5-mL subcutaneous injections in the right paramedial abdominal surface with either Matrigel alone or Matrigel plus 50 ng/mL basic fibroblast growth factor (bFGF) (Boehringer Mannheim).31 After injection, the mice were returned to their cages for normal feeding for 7 days, at which time all mice were killed. Matrigel plugs were quickly recovered with overlying skin or abdominal musculature for support and immediately fixed in 4% formaldehyde in phosphate buffer and 5% sucrose. The plugs were embedded in paraffin, sectioned, stained with Masson's trichrome, examined for ingrowth of blood vessels, and photographed. The number of cells invading the plug was quantified with the NIH IMAGE 1.47 processing and analysis program at x20 magnification. Total numbers of cells and of vWF-positive cells were determined and expressed as mean cell area per field ±SEM from 20 random fields for each data point. In each of four different experiments, four to five mice were used for each data point. Gross and histological examination at day 7 showed no evidence of inflammation in control or bFGF-containing Matrigel plugs.
Immunocytochemistry
Five-micrometer sections were prepared
from paraffin-embedded
Matrigel plugs. After deparaffinization and rehydration in PBS, the
sections were stained according to a streptavidin-biotin
immunoperoxidase technique (Histomark System, Kirkegaard and Perry).
The primary antibody, mouse monoclonal to human vWF (Boehringer
Mannheim) used at 2 µg/mL, was detected with biotinylated goat
anti-mouse IgG followed by streptavidin-horseradish peroxidase and
diaminobenzidine (Histostain-SP, Zymed Laboratories). Nonimmune rabbit
IgG at the same concentration was used as the negative control.
Data Analysis
Each experiment was performed at least three
times, with each
data point performed in triplicate. Results are expressed as mean±SEM.
Significance of results was determined with INSTAT 2.0
data analysis software for Macintosh (Graph Pad). Most comparisons
were performed with Student's t test for unpaired data,
unless significant differences in the SDs between two groups dictated
use of a nonparametric tool such as the Welch alternate t
test.
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Results |
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Effect of 17ß-Estradiol and Other Gonadal Steroids on HUVEC Adhesion to Matrix Proteins
Adherence of HUVECs to laminin was tested in hormone-free medium with 0.1 to 2 ng/mL testosterone, progesterone, or 17ß-estradiol. After 0.5 hour of incubation, a significantly greater increase in adherence was seen in the presence of estradiol. Although progesterone- and testosterone-treated endothelial cells also showed some enhanced attachment to laminin (Fig 1
), estradiol-treated cells
showed significantly greater enhancement at each dose tested. The
greatest adherence was observed when cells were incubated with
estradiol at 1.5 to 2 ng/mL. At all doses tested, HUVECs treated with
estradiol showed 25% to 35% greater attachment than did cells treated
with progesterone or testosterone.
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Similar estradiol-promoted increases
in adhesion were observed with
attachment to plastic and to other extracellular matrix proteins. As
shown in Fig 2, at 60 minutes, estradiol increased
adhesion by 20% to 35% when cells were allowed to attach to plastic
or to wells coated with laminin, type I collagen, type IV collagen, or
fibronectin. A time-course experiment was performed to determine
whether the effects of estradiol on adhesion were maximal at any time
from 30 to 120 minutes. HUVECs incubated with 1.5 ng/mL estradiol
showed 20% to 30% greater adherence at all time points up to 2 hours,
without showing a peak effect at any particular time (data not
shown).
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17ß-Estradiol and Cell Migration
During angiogenesis,
cells that have attached to matrix
subsequently migrate across or through the matrix. To evaluate cell
migration in vitro, two assays were used. A defined "wound" was
scraped across a HUVEC monolayer cultured in hormone-free medium on
plastic. Thymidine was added to cultures to prevent cell proliferation
from contributing to wound closure. Cells cultured in 2 ng/mL estradiol
achieved greater wound closure within 24 hours than did cells without
estradiol (Fig 3). Results at different doses of
estradiol were quantified with an image analysis program, and a
dose-response curve was generated (Fig 4). At each time
point, a slight increase in the rate of wound closure was noted in the
presence of 1 ng/mL estradiol. At 2 ng/mL or more, the increase was
extremely significant, with 2 ng/mL doubling the rate of wound closure
and 3 ng/mL facilitating three times the wound closure of the untreated
cell.
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In a second assay, the ability of estradiol to enhance migration
in a
Boyden chamber assay was tested. Migration of HUVECs through a filter
toward an ECGS stimulus was enhanced in a dose-dependent manner by
estradiol (Fig 5). HUVECs treated with 1 ng/mL estradiol
showed more than twice the migratory activity of untreated cells, and
cells treated with 2 ng/mL migrated at almost three times the rate of
untreated cells.
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Proliferation of HUVECs
HUVECs were cultured in hormone-free
medium in the absence or
presence of 17ß-estradiol. In preliminary experiments, it was
determined that the enhancing effect of estradiol on proliferation
peaked at 1 to 2 ng/mL, with higher doses being less effective in
increasing cell number. In subsequent experiments, HUVECs were treated
with 1.5 ng/mL estradiol, and the number of cells present was
evaluated daily for 5 days. Estradiol-treated cells showed increased
proliferation over a 5-day period compared with cells cultured without
estradiol (Fig 6). This effect was apparent by day 2
after plating, and by day 5 there were three times the number of cells
on plastic when cultured with estradiol compared with controls without
estradiol. Over the same time course, estrogen-treated HUVECs cultured
on laminin showed a more pronounced enhancement, with a greater than
fivefold increase.
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) or
presence (
) of 1.5 ng/mL 17ß-estradiol. Each day, triplicate
cultures were detached from the plate with trypsin-EDTA and the number
of cells present was quantified by Coulter counter. Standard errors
were <1.05x10-3 for all time points. Similar results
were obtained in four separate experiments. Comparing control with
estradiol cultures, P<.005 for plastic day 1 and day 2;
P<.0001 for all other points after day 0 on plastic and on
laminin.
Effect of 17ß-Estradiol on HUVEC Tube Formation on Matrigel
HUVECs incubated on a Matrigel substratum align and organize into
tubular structures with a central lumen. To examine the role of
estrogens in this process, HUVECs were cultured in hormone-free medium
on phenol-free Matrigel. Estradiol was found to enhance formation of
capillary-like tubes on Matrigel in a dose-dependent manner (Fig
7). Tube formation was maximal at 1 to 2 ng/mL estradiol
(Fig 8); twice as many tubes formed as in cultures
without estradiol. Although tube area at estradiol concentrations >2
ng/mL was still greater than in untreated wells, some decrease in
enhancement was noted.
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Effect of an Estrogen Receptor Antagonist on Endothelial Cell
Function
The results described above indicate that estradiol enhances
several in vitro endothelial cell activities that are relevant to
angiogenesis, including adhesion, proliferation, migration, and
organization. The specific estrogen receptor antagonist ICI 182,780 was
evaluated for its ability to abrogate estradiol enhancement of HUVEC
activity in the plate-wounding assay and in tube formation. In the
experiment shown in Fig 9, estradiol (4 ng/mL) caused
HUVECs to migrate into a wounded monolayer 2.5 times faster than cells
in the absence of estradiol. ICI 182,780 partially inhibited the
estrogen effect at 10-9 mol/L. At 10-8 mol/L
inhibitor, equimolar with the amount of estradiol present, there
was complete inhibition of the estrogen effect, with no difference in
migration between cultures with inhibitor and cultures without
estradiol. In the tube-forming assay, maximal enhancement occurs with
3x10-9 mol/L estradiol. As was the case with the
wounding
assay, the estrogen receptor antagonist by itself did not alter
responses in the absence of estradiol. However, it did prevent
enhancement of tube formation by estrogen (Table 1).
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Role of Estrogen in a Murine Angiogenesis Assay
These in
vitro results suggest that estradiol enhances angiogenic
behavior by endothelial cells. A murine angiogenesis assay was used to
determine whether this hormone enhances vessel formation in
vivo. Matrigel and bFGF, a well-characterized angiogenic
factor,32 33 34 were coinjected
subcutaneously into
ovariectomized mice. By gross inspection after 7 days, Matrigel plugs
with no added bFGF remained clear, with no visible vessels in any
animals. In contrast, plugs with bFGF from estrogen-replete mice were
bright pink. Tortuous vessels were apparent within the gel, which was
often surrounded by a superficial network of blood vessels as well. In
contrast, plugs from estrogen-deficient mice were pale, with few
visible blood vessels. Sham-operated mice had the same response as
estrogen-replete ovariectomized mice. By histology, few or no vessels
are seen in the absence of bFGF in the sham-operated animals (Fig
10). The neovascularization seen in sham-operated
animals is readily apparent when bFGF is present. Vascularization
is markedly decreased in ovariectomized mice unless they receive
estrogen replacement. Quantitative analysis of histological
sections revealed that plugs containing bFGF from estradiol-replete
mice consistently had a greater number of cells than did plugs from
mice with placebo. These same plugs also showed a greater number of
cells staining positive for vWF, an endothelial cell marker (Table
2), indicating that total cellularity of the plugs
reflected the degree of endothelial invasion. Quantitative analysis
of plugs from sham-operated (nonovariectomized) mice demonstrated total
cell numbers, as well as vWF-positive cell numbers, similar to those
found in estrogen-replete ovariectomized mice.
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Discussion |
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Several studies suggest a correlation between new vessel formation and estrogens. Cutaneous hemangiomas are more common in female than in male infants.35 The spider angiomas of pregnancy disappear postpartum,36 and spider lesions seen in patients with hepatic cirrhosis are associated with elevated levels of serum estradiol.37 An analysis of postsclerotherapy patient records showed more telangiectasia in patients receiving gonadal hormones during therapy.38 In DMBA (9,10-dimethyl-1,2-benzonthracene)-induced rat breast carcinoma, tumor regression is effected by decreased estrogen levels, which precipitate regression of estrogen-dependent endothelial cells, thus compromising the vascular supply of the tumor.39 These studies suggest that pathological vessel formation is associated with an effect of estrogens on endothelial cell angiogenic activity. In support of this hypothesis, it has been reported that partial and pure estrogen antagonists inhibit angiogenesis in the chick egg chorioallantoic membrane assay.40
In the present series of experiments, we have investigated the effects of the gonadal steroid 17ß-estradiol on HUVEC adhesion, migration, proliferation, and differentiation. Estradiol enhanced adhesion of HUVECs to various matrix proteins and to tissue culture plastic. It promoted migration in a wounded monolayer of cells, in which contact inhibition is eliminated, and in a Boyden chamber assay. In the presence of estradiol, HUVEC proliferation was increased. Estradiol also increased formation of capillary-like networks by HUVECs on Matrigel. Both proliferation and tube formation were maximal at 1 to 2 ng/mL estradiol, with responses decreasing somewhat at higher doses. Thus, 17ß-estradiol enhances multiple components of angiogenic activity in vitro. We also studied new blood vessel formation in vivo. Estrogen enhanced endothelial cell invasion and formation of blood vessels in subcutaneous Matrigel plugs containing bFGF. In summary, multiple assays of endothelial cell activity were augmented by estradiol.
The series of assays used here was designed to examine various aspects of angiogenic behavior. After disruption of the vessel wall, endothelial cells must adhere to matrices in the stromal space, migrate out from the original vascular structure, proliferate, and organize into new vascular structures. Each of these activities is modeled in part by one of our in vitro assays. Adhesion to matrix substrata, cell division on plastic or on laminin, and migration in a Boyden chamber or a wounded monolayer were all enhanced in the presence of estradiol. The ability of endothelial cells to form a network of tubular structures across the surface of a Matrigel substratum is a complex phenomenon that combines elements of attachment, migration, organization, and differentiation.41 Organization on Matrigel is also exhibited by other cell types such as salivary gland,42 mammary,43 renal tubular,44 or bone45 cells. Thus, this phenomenon is not specific for endothelial cells. However, in each of these model systems, the cells show subtle morphological or functional changes that are specific for their tissue of origin. Organization by endothelial cells, for example, requires both matrix metalloproteinase17 and plasminogen activator46 activity similar to that suggested to be important for angiogenesis in other experimental systems.47 48 Further, the complex organizational behavior of HUVECs on Matrigel models the type of coordinated activities required for angiogenesis by endothelial cells. Thus, although the in vitro Matrigel model does not represent true angiogenesis per se, it suggests that estradiol is important for many of the activities that contribute to vessel formation. In vivo studies of vascularization of a Matrigel plug strongly suggest that estradiol plays a role in angiogenesis. Since neovascularization does not occur when Matrigel alone is injected, the Matrigel appears to function mainly as a three-dimensional lattice to support bFGF-induced vessel formation in this assay.
The mechanism of action of estrogen on endothelial cells remains to be determined. In another system, estradiol alters the expression and distribution in breast cancer cells of proteins that regulate cell-cell and cell-matrix interactions.49 Studies from our laboratory indicate that estradiol enhances expression of mRNA for several endothelial cell leukocyte adhesion molecules13 and integrins.50 The observation that estradiol enhances mRNA transcription suggested that the estrogen effects are mediated by the estrogen receptor, which serves as a transcription factor. Support for this hypothesis was provided by the observation that the specific estrogen receptor antagonist ICI 182,780 abrogates estrogenic enhancement of migration and tube formation by HUVECs in vitro.
Estradiol may enhance activity by augmenting the actions of angiogenic
growth factors. Vascular endothelial cell growth factor production is
found in tissues noted for either high estrogenic content or abundant
estrogen responsiveness.51 52 Further, in the present
series of experiments, neovascularization is significantly decreased in
ovariectomized mice despite the presence of bFGF in the in vivo
angiogenesis assay. Thus, bFGF is required for angiogenesis, but its
effects are more pronounced in the presence of estradiol. These results
are consistent with our previous observation that TNF
is
a necessary stimulus for expression of certain adhesion molecules by
HUVECs but that endothelial cell responses to TNF are
greatly enhanced by estradiol.13 One mechanism by which
bFGF may enhance angiogenesis is to induce cell
migration.53 54 The means by which estradiol
increases
bFGF effects is under study by several investigators. A relation has
been suggested between the stimulatory effects of estrogens and
production of bFGF and its receptor.55 56 This
relation
could account for the enhanced migration seen in the Boyden chamber
assay. Alternatively, in the "wounding" assay, soluble growth
factors could be released from injured cells after scraping. If this
latter mechanism were paramount, estradiol would appear to be
augmenting the response to growth factors that are already present,
as may also be the case with the in vivo study in the present
series of experiments. Studies in progress in our laboratory suggest a
complex relation between estradiol and bFGF actions in vitro.
Generally, estradiol appears to potentiate effects of low doses of bFGF
(K.A.M., H.W.S., unpublished observations).
Regulation of angiogenic responses has considerable clinical relevance. For example, in tumor biology, quantification of blood vessel formation in breast cancer biopsies may provide an indication of future metastatic risk.57 Studies of tumors lacking estrogen receptors suggest that antiestrogens may inhibit growth of these tumors via an indirect effect of estrogen involving new vessel formation.58 Angiogenesis is also important for the response to injury. Growth factors may play a major role in this process, and studies of interactions between growth factors and estradiol or antiestrogens may prove useful in optimizing control of angiogenesis. Thus, treatment of cardiovascular injury, diabetic complications, and vasculitis should be facilitated by further understanding of angiogenic activity. The studies of the effect of estradiol on angiogenic behavior described here indicate one potential mechanism regulating this activity.
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Acknowledgments |
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This study was supported in part by grant HL-53918 from the National Heart, Lung, and Blood Institute. Dr Morales and Ms McGowan are Howard Hughes Medical Institute–National Institutes of Health Research Scholars. We appreciate proviion of the estrogen receptor antagonist ICI 182,780 by Zeneca Pharmaceuticals.
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Footnotes |
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Reprint requests to H. William Schnaper, MD, Pediatrics W-140, Northwestern University Medical School, 303 E Chicago Ave, Chicago, IL 60611.
Received June 27, 1994; accepted September 23, 1994.
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References |
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TopAbstractIntroductionMethods Results Discussion References |
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1. Findlay JK. Angiogenesis in reproductive tissues. J Endocrinol. 1986;111:357-366.
2. Lahita RG. Sex steroids and the rheumatic diseases. Arth Rheum. 1985;28:121-126. [Medline] [Order article via Infotrieve]
3. Engerman RL. Pathogenesis of diabetic retinopathy. Diabetes. 1989;38:1203-1206. [Abstract]
4. Hunt TK. Wound Healing and Wound Infection: Theory and Surgical Practice. New York, NY: Appleton-Century-Crofts; 1980.
5. Madden JW, Arem AJ. Wound healing: biological and clinical features. In: Sabiston DC, ed. Textbook of Surgery. Philadelphia, Pa: WB Saunders; 1991:164-175.
6.
Folkman J. What is the evidence that tumors are angiogenesis
dependent? J Natl Cancer Inst. 1990;82:4-6.
7. Ausprunk DH, Folkman J. Migration and proliferation of endothelial cells in preformed and newly formed vessels during tumor angiogenesis. Microvasc Res. 1977;14:53-65. [Medline] [Order article via Infotrieve]
8. Furcht LT. Critical factors in controlling angiogenesis: cell products, cell matrix, and growth factors. Lab Invest. 1986;55:505-509. [Medline] [Order article via Infotrieve]
9. Grossman C. Possible underlying mechanisms of sexual dimorphism in the immune response: fact and hypothesis. J Steroid Biochem. 1989;34:241-251. [Medline] [Order article via Infotrieve]
10. Shelhamer JH, Volkman DJ, Parillo JE, Lawley TJ, Johnston MR, Fauci AS. Takayasu's arteritis and its therapy. Ann Intern Med. 1985;103:121-126.
11. Ahmed SA, Penhale WJ, Talal N. Sex hormones, immune responses, and autoimmune diseases: mechanisms of sex hormone action. Am J Pathol. 1985;121:531-551. [Abstract]
12. Cutolo M, Balleari E, Giusti M, Intra E, Accardo S. Androgen replacement therapy in male patients with rheumatoid arthritis. Arth Rheum. 1991;34:1-5. [Medline] [Order article via Infotrieve]
13. Cid MC, Kleinman HK, Grant DS, Schnaper HW, Fauci AS, Hoffman GS. Estradiol enhances leukocyte binding to tumor necrosis factor (TNF)-stimulated endothelial cells via an increase in TNF-induced adhesion molecules E-selectin, intercellular adhesion molecule type 1 and vascular cell adhesion molecule type 1. J Clin Invest. 1994;93:17-25.
14. Torry RJ, Rongish BJ. Angiogenesis in the uterus: potential regulation and relation to tumor angiogenesis. Am J Reprod Immunol. 1992;27:171-179.
15. Simon J, Rodi I, Stumpf P, Sauer M, Cohen S, Ford L, Bustillo M, Buster J. Polysiloxane vaginal rings and cylinders for physiologic endometrial priming in functionally agonadal women. Fertil Steril. 1986;46:619-625. [Medline] [Order article via Infotrieve]
16. Sauer M, Rodi I, Simon J, Buster J. Scanning electron microscopy of the endometrium in functionally agonadal women receiving steroid replacement therapy with polysiloxane vaginal rings and cylinders. J Reprod Med. 1987;32:688-692. [Medline] [Order article via Infotrieve]
17. Schnaper HW, Grant DS, Stetler-Stevenson WG, Fridman R, D'Orazi G, Murphy A, Bird RE, Hoyhtya M, Fuerst TR, Quigley J, French D, Kleinman HK. Type IV collagenases and TIMPs modulate endothelial cell morphogenesis in vitro. J Cell Physiol. 1993;156:235-246.
18. Amberger A, Bauer H, Tontsch U, Gabbiani G, Kocher O, Bauer HC. Reversible expression of sm alpha-actin protein and sm alpha-actin mRNA in cloned cerebral endothelial cells. FEBS Lett. 1991;287:223-225. [Medline] [Order article via Infotrieve]
19. Folkman J, Haudenschild C. Angiogenesis in vitro. Nature. 1980;288:551-556. [Medline] [Order article via Infotrieve]
20.
Wagner DD, Marder VJ. Biosynthesis of von Willebrand protein
by human endothelial cells: processing steps and their intracellular
location. J Cell Biol. 1984;99:2123-2130.
21.
Thompson EW, Reich R, Shima TB, Albini A, Graf J, Martin GR,
Dickson RB, Lippman ME. Differential regulation of growth and
invasiveness of MCF-7 breast cancer cells by antiestrogens.
Cancer Res. 1988;48:6764-6768.
22. Thompson EW, Katz D, Shima TB, Wakeling AE, Lippman ME, Dickson RB. ICI 164,384, a pure antagonist of estrogen-stimulated MCF-7 cell proliferation and invasiveness. Cancer Res. 1989; 49:6929-6934.
23. Bakerman S. ABC's of Interpretive Laboratory Data. 2nd ed. Greenville, NC: Interpretive Laboratory Data, Inc; 1984.
24. Klebe RJ. Isolation of a collagen-dependent cell attachment factor. Nature. 1974;250:248-251. [Medline] [Order article via Infotrieve]
25. Timpl R, Rohde H, Robey PG, Remand SI, Foidart JM, Martin GR. Laminin-A glycoprotein from basement membranes. J Biol Chem. 1979;254:9932-9933.
26. Kleinman HK. Isolation and characterization of type IV procollagen, laminin and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry. 1982;24:6188-6193.
27. Grant DS, Tashiro K-I, Segui-Real B, Yamada Y, Martin GR, Kleinman HK. Two different domains of laminin mediate the differentiation of human endothelial cells into capillary-like structures in vitro. Cell. 1989;58:933-943. [Medline] [Order article via Infotrieve]
28. Pepper MS, Sappino AP, Montesano R, Orci L, Vassali J-D. Plasminogen activator inhibitor-1 is induced in migrating endothelial cells. J Cell Physiol. 1992;153:129-139. [Medline] [Order article via Infotrieve]
29. Albini A, Mitchell CD, Thompson EW, Seeman R, Martin GR, Wittek AE, Quinnan GV. Invasive activity and chemotactic response to growth factors by Kaposi's sarcoma cells. J Cell Biochem. 1988;36:369-376. [Medline] [Order article via Infotrieve]
30. Kleinman HK, McGarvey ML, Hassell JR, Star VL, Cannon FB, Laurie GW, Martin GR. Basement membrane complexes with biological activity. Biochemistry. 1986;25:312-318. [Medline] [Order article via Infotrieve]
31. Passaniti A, Taylor RM, Pili R, Guo Y, Long PV, Haney JA, Pauly RR, Grant DS, Martin GR. A simple quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin and fibroblast growth factor. Lab Invest. 1992;67:519-528. [Medline] [Order article via Infotrieve]
32. Reidy MA, Lindner V. Basic FGF and growth of arterial cells. Ann N Y Acad Sci. 1991;638:290-299. [Medline] [Order article via Infotrieve]
33.
Gospodarowicz D, Ferrara N, Schweigerer L, Neufeld G.
Structural characterization and biological functions of fibroblast
growth factor. Endocr Rev. 1987;8:95-114.
34.
Folkman J, Klagsbrun M. Angiogenic factors.
Science. 1987;235:442-447.
35.
Bowers RE, Graham EA, Tomlinson KM. The natural history of the
strawberry nevus. AMA Arch Dermatol. 1960;82:667-680.
36. Corbett DA. Discussion. Br J Dermatol. 1914;26:200.
37. Pirovino M, Linder R, Boss CH. Cutaneous spider nevi in liver cirrhosis: capillary microscopical and hormonal investigation. Klin Wochenschr. 1988;66:298-302. [Medline] [Order article via Infotrieve]
38. Davis LT, Duffy DM. Determination of incidence and risk factors for postsclerotherapy telangiectatic matting of the lower extremity: a retrospective analysis. J Dermatol Surg Oncol. 1990;16:327-330. [Medline] [Order article via Infotrieve]
39. Fukuda M, Maefawa J, Hosokawa Y, Urata Y, Sugihara H, Hattori T, Miyoshi N, Nakanishi K, Fujita S. Hormone-dependent changes of blood vessels in DMBA-induced rat mammary carcinoma and its regression studied by 3H-thymidine autoradiography. Basic Appl Histochem. 1985;29:21-43. [Medline] [Order article via Infotrieve]
40.
Gagliardi A, Collins DC. Inhibition of angiogenesis by
antiestrogens. Cancer Res. 1993;53:533-535.
41. Grant DS, Lelkes PI, Fukuda K, Kleinman HK. Intracellular mechanisms involved in basement membrane induced blood vessel differentiation in vitro. In Vitro Cell Devel Biol. 1991;27A:327-336.
42. Kibbey MC, Royce LS, Dym MS, Baum BJ, Kleinman HK. Glandular morphogenesis of a human submandibular cell line by basement membrane components in vitro. Exp Cell Res. 1992;198:343-357. [Medline] [Order article via Infotrieve]
43. Seely KA, Aggeler J. Modulation of milk protein synthesis through alteration of the cytoskeleton in mouse mammary epithelial cells cultured on a reconstituted basement membrane. J Cell Physiol. 1991;146:117-130. [Medline] [Order article via Infotrieve]
44.
Taub M, Wang Y, Szcesney TM, Kleinman HK, Martin GR.
Transforming growth factor alpha is required for kidney tubulogenesis
in Matrigel cultures in serum-free medium. Proc Natl Acad Sci
U S A. 1990;87:4002-4006.
45. Vukicevic S, Luyten FP, Kleinman HK, Reddi AH. Differentiation of canaliculi-like bone cell network by basement membrane components and defined domains of laminin. Cell. 1990;63:437-445. [Medline] [Order article via Infotrieve]
46. Schnaper HW, Barnathan ES, Kleinman HK. Plasminogen activator regulation during endothelial cell morphogenesis in vitro. Pediatr Res. 1993;33:56A. Abstract.
47.
Pepper MS, Vassalli JD, Montesano R, Orci L. Urokinase-type
plasminogen activator is induced in migrating capillary endothelial
cells. J Cell Biol. 1987;105:2535-2541.
48.
Moses MA, Sudhalter J, Langer R. Identification of an
inhibitor of neovascularization from cartilage. Science. 1990;248:1408-1410.
49. DePasquale JA, Samsonoff WA, Gierthy JF. 17-ß-Estradiol induced alterations of cell-matrix and intercellular adhesions in a human mammary carcinoma line. J Cell Sci. 1994;107:1241-1254. [Abstract]
50. Cid MC, Esparza J, Grant DG, Morales DE, McGowan KA, Urbano-Marquez A, Schnaper HW, Kleinman HK. Estradiol increases endothelial cell attachment to extracellular matrixproteins through an increase in integrin expression. Clin Res. 1994;42:131A. Abstract.
51. Shweiki D, Itin A, Neufeld G, Gitay-Goren H, Keshet E. Patterns of expression of vascular endothelial growth factor (VEGF) and VEGF receptors in mice suggest a role in hormonally regulated angiogenesis. J Clin Invest. 1993;91:2235-2243.
52. Cullinan-Bove K, Koos RD. Vascular endothelial growth factor/vascular permeability factor expression in the rat uterus: rapid stimulation by estrogen correlates with estrogen-induced increases in uterine capillary permeability and growth. Endocrinology. 1993; 133:829-837.
53.
Mignatti P, Morimoto T, Rifkin DB. Basic fibroblast
growth factor released by single, isolated cells stimulates their
migration in an autocrine manner. Proc Natl Acad Sci
U S A. 1991;88:11007-11011.
54. Madri JA, Bell L, Marx M, Merwin JR, Basson C, Prinz C. Effects of soluble factors and extracellular matrix components on vascular cell behavior in vitro and in vivo: models of deendothelialization and repair. J Cell Biochem. 1991;45:123-130. [Medline] [Order article via Infotrieve]
55. Cullinan K, Koos R. Expression of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and keratinocyte growth factor in the rat uterus: stimulation by estrogen. Biol Reprod. 1991;44(suppl 1):A536. Abstract.
56. Lehtola L, Partanen J, Sistonen L, Korhonen J, Warri A, Harkonen P, Clarke R, Alitalo K. Analysis of tyrosine kinase mRNAs including four FGF receptor mRNAs expressed in MCF-7 breast-cancer cells. Int J Cancer. 1992;50:598-603. [Medline] [Order article via Infotrieve]
57. Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis: correlation in invasive breast carcinoma. N Engl J Med. 1991;324:1-8. [Abstract]
58. Cocconi G, Bella M, Calabresi F, Tonato M, Canaletti R, Boni C, Buzzi F, Ceci G, Gorgna F, Costa P, et al. Treatment of metastatic malignant melanoma with dacarbazine plus tamoxifen. N Engl J Med. 1992;327:516-522.[Abstract]
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|
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|
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|
|
|
|
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|
|
|
|
 |
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|
|
|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
 |
|
 A. Saitta, D. Altavilla, D. Cucinotta, N. Morabito, N. Frisina, F. Corrado, R. D'Anna, A. Lasco, G. Squadrito, A. Gaudio, et al. Randomized, Double-Blind, Placebo-Controlled Study on Effects of Raloxifene and Hormone Replacement Therapy on Plasma NO Concentrations, Endothelin-1 Levels, and Endothelium-Dependent Vasodilation in Postmenopausal Women Arterioscler. Thromb. Vasc. Biol., September 1, 2001; 21(9): 1512 - 1519. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Currie, S. P. Gunningham, C. Han, P. A. E. Scott, B. A. Robinson, A. L. Harris, and S. B. Fox Angiopoietin-1 Is Inversely Related to Thymidine Phosphorylase Expression in Human Breast Cancer, Indicating a Role in Vascular Remodeling Clin. Cancer Res., April 1, 2001; 7(4): 918 - 927. [Abstract] [Full Text]
|
|
|
|
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 W. Oitzinger, R. Hofer-Warbinek, J. A. Schmid, Y. Koshelnick, B. R. Binder, and R. de Martin Adenovirus-mediated expression of a mutant I{kappa}B kinase 2 inhibits the response of endothelial cells to inflammatory stimuli Blood, March 15, 2001; 97(6): 1611 - 1617. [Abstract] [Full Text] [PDF]
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 R. K. Dubey and E. K. Jackson Estrogen-induced cardiorenal protection: potential cellular, biochemical, and molecular mechanisms Am J Physiol Renal Physiol, March 1, 2001; 280(3): F365 - F388. [Abstract] [Full Text] [PDF]
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Z. S. Kyriakides, P. Petinakis, L. Kaklamanis, E. Sbarouni, P. Karayannakos, D. Iliopoulos, I. Dontas, and D. Th. Kremastinos Intramuscular administration of estrogen may promote angiogenesis and perfusion in a rabbit model of chronic limb ischemia Cardiovasc Res, February 16, 2001; 49(3): 626 - 633. [Abstract] [Full Text] [PDF]
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D. W. Losordo and J. M. Isner Estrogen and Angiogenesis : A Review Arterioscler. Thromb. Vasc. Biol., January 1, 2001; 21(1): 6 - 12. [Abstract] [Full Text] [PDF]
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M. Bonenfant, C. H. Blomquist, P. R. Provost, R. Drolet, P. D’Ascoli, and Y. Tremblay Tissue- and Site-Specific Gene Expression of Type 2 17{beta}-Hydroxysteroid Dehydrogenase: In Situ Hybridization and Specific Enzymatic Activity Studies in Human Placental Endothelial Cells of the Arterial System J. Clin. Endocrinol. Metab., December 1, 2000; 85(12): 4841 - 4850. [Abstract] [Full Text]
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P. Bausero, M.-H. Ben-Mahdi, J.-P. Mazucatelli, C. Bloy, and M. Perrot-Applanat Vascular endothelial growth factor is modulated in vascular muscle cells by estradiol, tamoxifen, and hypoxia Am J Physiol Heart Circ Physiol, November 1, 2000; 279(5): H2033 - H2042. [Abstract] [Full Text] [PDF]
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T. Hayashi, M. Jayachandran, D. Sumi, N. K. Thakur, T. Esaki, E. Muto, H. Kano, Y. Asai, and A. Iguchi Physiological Concentration of 17{beta}-Estradiol Retards the Progression of Severe Atherosclerosis Induced by a High-Cholesterol Diet Plus Balloon Catheter Injury : Role of NO Arterioscler. Thromb. Vasc. Biol., June 1, 2000; 20(6): 1613 - 1621. [Abstract] [Full Text] [PDF]
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 L. Wroblewski Lissin and J. P. Cooke Phytoestrogens and cardiovascular health J. Am. Coll. Cardiol., May 1, 2000; 35(6): 1403 - 1410. [Abstract] [Full Text] [PDF]
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S.-H. Yang, J. Shi, A. L. Day, J. W. Simpkins, and S. E. Robinson Estradiol Exerts Neuroprotective Effects When Administered After Ischemic Insult • Editorial Comment Stroke, March 1, 2000; 31(3): 745 - 750. [Abstract] [Full Text] [PDF]
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 T. A. Parker, D. Dunbar Ivy, H. L. Galan, T. R. Grover, J. P. Kinsella, and S. H. Abman Estradiol improves pulmonary hemodynamics and vascular remodeling in perinatal pulmonary hypertension Am J Physiol Lung Cell Mol Physiol, February 1, 2000; 278(2): L374 - L381. [Abstract] [Full Text] [PDF]
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 R. H. Karas, J. B. Hodgin, M. Kwoun, J. H. Krege, M. Aronovitz, W. Mackey, J. A. Gustafsson, K. S. Korach, O. Smithies, and M. E. Mendelsohn Estrogen inhibits the vascular injury response in estrogen receptor beta -deficient female mice PNAS, December 21, 1999; 96(26): 15133 - 15136. [Abstract] [Full Text] [PDF]
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E. Tan, M. V Gurjar, R. V Sharma, and R. C Bhalla Estrogen receptor-{alpha} gene transfer into bovine aortic endothelial cells induces eNOS gene expression and inhibits cell migration Cardiovasc Res, August 15, 1999; 43(3): 788 - 797. [Abstract] [Full Text] [PDF]
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 I. Suzuma, M. Mandai, H. Takagi, K. Suzuma, A. Otani, H. Oh, K. Kobayashi, and Y. Honda 17 {beta}-Estradiol Increases VEGF Receptor-2 and Promotes DNA Synthesis in Retinal Microvascular Endothelial Cells Invest. Ophthalmol. Vis. Sci., August 1, 1999; 40(9): 2122 - 2129. [Abstract] [Full Text] [PDF]
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N. Harada, H. Sasano, H. Murakami, T. Ohkuma, H. Nagura, and Y. Takagi Localized Expression of Aromatase in Human Vascular Tissues Circ. Res., June 11, 1999; 84(11): 1285 - 1291. [Abstract] [Full Text] [PDF]
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M. E. Mendelsohn and R. H. Karas The Protective Effects of Estrogen on the Cardiovascular System N. Engl. J. Med., June 10, 1999; 340(23): 1801 - 1811. [Full Text] [PDF]
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T. L. Moser, M. S. Stack, I. Asplin, J. J. Enghild, P. Hojrup, L. Everitt, S. Hubchak, H. W. Schnaper, and S. V. Pizzo Angiostatin binds ATP synthase on the surface of human endothelial cells PNAS, March 16, 1999; 96(6): 2811 - 2816. [Abstract] [Full Text] [PDF]
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S. W. McLeskey, C. A. Tobias, P. R. Vezza, A. C. Filie, F. G. Kern, and J. Hanfelt Tumor Growth of FGF or VEGF Transfected MCF-7 Breast Carcinoma Cells Correlates with Density of Specific Microvessels Independent of the Transfected Angiogenic Factor Am. J. Pathol., December 1, 1998; 153(6): 1993 - 2006. [Abstract] [Full Text] [PDF]
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S. Kim-Schulze, W. L. Lowe Jr, and H. W. Schnaper Estrogen Stimulates Delayed Mitogen-Activated Protein Kinase Activity in Human Endothelial Cells via an Autocrine Loop That Involves Basic Fibroblast Growth Factor Circulation, August 4, 1998; 98(5): 413 - 421. [Abstract] [Full Text] [PDF]
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M. Lansink, P. Koolwijk, V. van Hinsbergh, and T. Kooistra Effect of Steroid Hormones and Retinoids on the Formation of Capillary-Like Tubular Structures of Human Microvascular Endothelial Cells in Fibrin Matrices Is Related to Urokinase Expression Blood, August 1, 1998; 92(3): 927 - 938. [Abstract] [Full Text] [PDF]
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E. Van Belle, C. Bauters, T. Asahara, and J. M. Isner Endothelial regrowth after arterial injury: from vascular repair to therapeutics Cardiovasc Res, April 1, 1998; 38(1): 54 - 68. [Full Text] [PDF]
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N. J. Alkayed, I. Harukuni, A. S. Kimes, E. D. London, R. J. Traystman, P. D. Hurn, and P. A. Grady Gender-Linked Brain Injury in Experimental Stroke • Editorial Comment Stroke, January 1, 1998; 29(1): 159 - 166. [Abstract] [Full Text] [PDF]
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Y. S. Schiffenbauer, R. Abramovitch, G. Meir, N. Nevo, M. Holzinger, A. Itin, E. Keshet, and M. Neeman Loss of ovarian function promotes angiogenesis in human ovarian carcinoma PNAS, November 25, 1997; 94(24): 13203 - 13208. [Abstract] [Full Text] [PDF]
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V. L. Baker, V. A. Chao, J. T. Murai, C. J. Zaloudek, and R. N. Taylor Human Umbilical Vessels and Cultured Umbilical Vein Endothelial and Smooth Muscle Cells Lack Detectable Protein and mRNA Endocing Estrogen Receptors Reproductive Sciences, November 1, 1997; 4(6): 316 - 324. [Abstract] [PDF]
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K. Krasinski, I. Spyridopoulos, T. Asahara, R. van der Zee, J. M. Isner, and D. W. Losordo Estradiol Accelerates Functional Endothelial Recovery After Arterial Injury Circulation, April 1, 1997; 95(7): 1768 - 1772. [Abstract] [Full Text]
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I. Spyridopoulos, A. B. Sullivan, M. Kearney, J. M. Isner, and D. W. Losordo Estrogen-Receptor–Mediated Inhibition of Human Endothelial Cell Apoptosis : Estradiol as a Survival Factor Circulation, March 18, 1997; 95(6): 1505 - 1514. [Abstract] [Full Text]
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S. Kim-Schulze, K. A. McGowan, S. C. Hubchak, M. C. Cid, M. B. Martin, H. K. Kleinman, G. L. Greene, and H. W. Schnaper Expression of an Estrogen Receptor by Human Coronary Artery and Umbilical Vein Endothelial Cells Circulation, September 15, 1996; 94(6): 1402 - 1407. [Abstract] [Full Text]
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C. D. Venkov, A. B. Rankin, and D. E. Vaughan Identification of Authentic Estrogen Receptor in Cultured Endothelial Cells: A Potential Mechanism for Steroid Hormone Regulation of Endothelial Function Circulation, August 15, 1996; 94(4): 727 - 733. [Abstract] [Full Text]
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V. Guetta and R. O. Cannon III Cardiovascular Effects of Estrogen and Lipid-Lowering Therapies in Postmenopausal Women Circulation, May 15, 1996; 93(10): 1928 - 1937. [Full Text]
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M. Gerhard and P. Ganz How Do We Explain the Clinical Benefits of Estrogen? : From Bedside to Bench Circulation, July 1, 1995; 92(1): 5 - 8. [Full Text]
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D. Pratico and G. A. FitzGerald Testosterone and Thromboxane : Of Muscles, Mice, and Men Circulation, June 1, 1995; 91(11): 2694 - 2698. [Full Text]
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M. Razandi, A. Pedram, and E. R. Levin Estrogen Signals to the Preservation of Endothelial Cell Form and Function J. Biol. Chem., December 1, 2000; 275(49): 38540 - 38546. [Abstract] [Full Text] [PDF]
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