From the Department of Pathology, Beth
Israel-Deaconess Medical Center and Harvard Medical School,
Boston, Massachusetts 02215, the ¶ Department of Cell Biology,
Baylor College of Medicine, Houston, Texas 77030, and the
Department of Pathology, University of Colorado Health Sciences
Center, Denver, Colorado 80262
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ABSTRACT |
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The use of steroid hormones in postmenopausal
replacement therapy has been associated with prevention of
cardiovascular disease. Although the contribution of estradiol to
endothelial cell function has been addressed, little information is
available on the effect of progestins on this cell type. Here, we
provide direct evidence for the presence of functional nuclear
progesterone receptor in endothelial cells and demonstrate that
physiological levels of progesterone inhibit proliferation through a
nuclear receptor-mediated mechanism. The effects of progesterone were
blocked by pretreatment with a progesterone receptor antagonist, and
progesterone receptor-deficient endothelial cells failed to respond to
the hormone. We evaluated the effect of progesterone by analysis of
aorta re-endothelialization experiments in wild-type and progesterone
receptor knockout mice. The rate of re-endothelialization was
significantly decreased in wild-type mice when in the presence of
progesterone, whereas there was no difference between control and
progesterone-treated progesterone receptor knockout mice. FACS analysis
showed that progestins arrest endothelial cell cycle in
G1. The lag in cell cycle progression involved
reduction in cyclin-dependent kinase activity, as shown by
down-regulation in retinoblastoma protein phosphorylation. In addition,
treatment of endothelial cells with progestins altered the expression
of cyclin E and A in accordance with G1 arrest. These
results have important implications to our current knowledge of the
effect of steroids on endothelial cell function and to the overall
contribution of progesterone to vascular repair.
Progesterone receptor
(PR)1 is a member of a family
of nuclear receptors capable of regulating gene expression upon binding to the appropriate hormones (1-4). As such, PR is considered a
transcription factor with known ability to influence development and
morphogenesis (5). Aside from its expression in several cell types of
the mammary gland, uterus, and ovary, PR has also been identified in
brain and vascular tissue (6-8). The presence of PR in blood vessels
has relevance because of the increase use of progestins in hormone
replacement therapy (HRT).
HRT with estrogen and progestins has become a well accepted treatment
regimen for postmenopausal women, particularly those with an absence of
familial breast cancer history. A large body of literature has
demonstrated that the benefits of HRT extend beyond the amelioration of
symptoms associated with menopause. HRT also aids in the prevention of
osteoporosis and in the reduction of cardiovascular disease (9-11).
Most of these effects have been attributed to estradiol, as
demonstrated by several experimental and epidemiological studies. In
fact, estradiol has been shown to diminish the incidence of
cardiovascular disease up to 45% (12-14). The contribution of
progesterone to the overall effect of HRT is less clear. Historically,
progestins were included to counteract endometrial dysplasia caused by
estradiol (11, 15). Although the inclusion of progestins to HRT has
recognized value, dosage levels and frequency have been the object of
much controversy, mostly due to collateral effects (16-18).
In blood vessels, PR has been localized in smooth muscle cells of the
tunica media (7, 19, 20) and has been shown to suppress smooth muscle
cell proliferation in vitro (8). Nevertheless, neither the
presence of functional PR nor the effect of progesterone have been
addressed on endothelial cells. Because constant levels of exogenous
progestins can be associated with the breakdown of vessels and
irregular endometrial bleedings (21), we hypothesized that this hormone
might play a direct role in endothelial function. Because progesterone
mediates signals through its receptor (2-4), we investigated the
presence of PR in endothelial cells from several organs and addressed
the effect of the ligand on several aspects of endothelial function.
Immunohistochemistry--
A mouse monoclonal antibody (1294) was
produced against purified full-length recombinant human progesterone
receptor. Monoclonal antibody 1294 recognizes an epitope in the amino
terminus located between amino acids 165 and 534 (22) and specifically
reacts with both the A and B isoforms of human PR as determined by
immunoprecipitation and Western blot of receptor positive (T47D) and
negative cells (MDA-231). Nuclear localization of receptor has been
observed by immunocytochemistry in female reproductive tissues and in
tumors of reproductive
glands.2 Specimens from
normal and atherosclerotic human arteries were obtained from the
Division of Surgical Pathology at Beth Israel Deaconess Medical Center,
Boston, MA. Tissue was fixed in 4% paraformaldehyde, embedded in
paraffin, and then sectioned at 5 µm. Sections were cleared,
hydrated, and blocked as described previously (23), and subsequently
incubated with anti-progesterone receptor monoclonal antibody 1294 (8.5 µg/ml) and polyclonal antibody to vWF (Dako, Carpinteria, CA)
followed by Texas Red-conjugated anti-mouse IgG (5 µg/ml) (Vector
Laboratories, Burlingame, CA) and anti-rabbit biotinylated (3 µg/ml)
(Vector Laboratories). Finally, avidin-fluorescein isothiocyanate was
applied, and tissues were incubated with Hoechst 33258 (10 µg/ml in
phosphate-buffered saline) (Molecular Probes; Eugene, OR) for nuclear
counterstaining. Controls included incubation with mouse IgG instead of
primary antibody.
Cell Culture--
Human dermal endothelial cells (HDECs) and
human endometrial endothelial cells were isolated as described
previously (24, 25). Mouse brain endothelial cells were harvested from
adult mice by enzymatic dissociation and further purified using
anti-PECAM (Pharmingen, San Diego, CA) linked to magnetic beads (Dynal,
Lake Succes, NY). The purity and homogeneity of cultures isolated in our laboratory was evaluated by uptake of acetylated low density lipoprotein, presence of PECAM, CD-34, up-regulation of E-selectin and
proliferation in response to VEGF (25). Human coronary endothelial cells and human lung endothelial cells were obtained from Clonetics (San Diego, CA). All cell types were grown in dishes precoated with
Vitrogen (Collagen Corp., Palo Alto, CA) and cultured in EBM
(Clonetics) supplemented with 15% fetal calf serum (FCS), 25 µg/ml
cAMP, and 1 µg/ml hydrocortisone-21-acetate and were used for
passages 3-6. Cells were made quiescent by incubation of confluent
monolayers with phenol red and serum-free EBM containing 0.2% bovine
serum albumin for 48 h. Experiments in which steroid function was
assessed were also performed in phenol red and serum-free EBM or in the
presence of charcoal-filtered serum. Progesterone was obtained from
Sigma; R5020 (17a,21-dimethyl-19-norpregna-4,9-dieme-3,20-one) from NEN
Life Science Products; and RU486 (17a-hydroxy-11
[4-dimethyl-aminophenyl] 17-propenyl-estra-4,5-dieme-3-one) was a
gift from Roussel-UCLAF (Romainville, France).
Northern Analysis--
Total RNA from cultured cells was
isolated as described previously (26). Purification of poly(A)+ RNA
from 100 µg of total RNA was performed by oligo(dT) binding to
magnetic beads following the manufacturer's specifications (Boehringer
Mannheim). mRNA was resolved on a 1% agarose gel, transferred to
nylon membranes, and hybridized to a 32P-labeled cDNA
fragment that included the entire reading frame of the PR. A 36B4 probe
was used to control for loading and transfer efficiency.
Immunoprecipitation of Progesterone Receptor--
Cultures of
HDECs and T47D (as positive control) were harvested with lysis buffer
(radioimmune precipitation buffer: 1× phosphate-buffered saline, 1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml PMSF).
Cell extracts were quantified using the DC protein assay (Bio-Rad).
Lysates (5 µg from T47D and 200 µg from endothelial cultures) were
incubated with anti-PR monoclonal antibody (1294) overnight at 4 °C.
Immunocomplexes were captured with a rabbit anti-mouse IgG linked to
Sepharose beads. After several washes in radioimmune precipitation
buffer, beads were boiled, and the supernatant was subjected to
separation by SDS-polyacrylamide gel electrophoresis. Further Western
analysis were performed with the same primary antibody used for
immunoprecipitation. Detection of immunocomplexes was done using the
ECL system (Amersham Pharmacia Biotech).
Transactivation Assays--
Transactivation mediated by the
progesterone receptor was evaluated by transfection with a plasmid
(pPRE) containing six tandem repeats of the progesterone responsive
element. Additional plasmids used as controls included: pCMV-Renilla
(as co-transfection control) and pPRE-scramble, the same plasmid as
pPRE, but with a scramble sequence that does not allow binding to PR.
Transfections were performed on the following cell types: endometrial
stromal cells, human endometrial endothelial cells, human umbilical
vein endothelial cells, human lung endothelial cells, human coronary
endothelial cells, HDECs, and bovine aortic endothelial cells. Cells
were plated onto 60-mm2 dishes at 80-90% confluence.
Prior to transfections, cells were washed and preincubated for 1 h
in serum and phenol red-free medium. Plasmid DNA was introduced into
cells by addition to cell cultures with the compound LipofectAMINETM
(Life Technologies, Inc.). Cell layers were given fresh medium after
6 h of transfection. After 24 h, cultures were treated with
either vehicle or 0.1 µM progesterone for 6-12 h. Cell
layers were harvested for assessment of renilla and luciferase
induction following the manufacturer's recommendations (Promega,
Madison, WI). Co-transfection efficiency and total amount of protein
were used to normalize transactivation of luciferase, as described
previously (27).
Evaluation of Progesterone Receptor Binding--
Cytosolic
extractions of HDECs in triplicate from early and late passage
cultures, as well as human endometrial stromal cells (as a positive
control), were subjected to incubation with increasing concentrations
(0.2-30 nM) of
[17- Proliferation Assays--
Quiescent HDECs were trypsinized and
plated onto 24-well dishes in phenol red-free EBM supplemented with 1%
FCS and 50 ng/ml VEGF (Pepro Technology Inc., Rocky Hill, NJ), in the
presence of either progesterone, R5020, RU486, or vehicle control.
During the last 4-8 h of each time point, cells were pulsed with 1 µCi/well of [3H]thymidine (NEN Life Science Products).
Incorporated [3H]thymidine was measured as described
previously (28).
Re-endothelialization Assays--
Aortae from wild-type and PRKO
mice were dissected and subsequently injured with the aid of mechanical
manipulators. Injury was performed so that only the endothelium was
removed, with minimal damage transmitted to the underlying smooth
muscle layers; this was ensured by providing constant pressure during
injury. After injury, aortae were stained with a solution of 0.01%
Evans blue; this dye binds to areas that are devoid of endothelium. The
blue stained areas indicated the extension of the injury at day 0. Aortae were attached on the surface of paraffin-coated plates with the
aid of thin needles and were cultured in EBM supplemented with 1% FCS
and 2 ng/ml FGF-2 (a generous gift from Dr. Gera Neufeld, Israel
Institute of Technology, Haifa, Israel) in the presence or absence of 1 µM progesterone during 4 days at 37 °C. At the end of
treatment (day 4), specimens were restained with Evans blue, and
extension of re-endothelialization was evaluated by measurements of the
injured area at days 0 and 4. The advantage of this procedure over
injury in vivo is that it enables to evaluate the
progression of the re-endothelialization over time and compare it to
the extent of the injury at day 0 in a quantitative manner. To compare
data groups, values were converted to percentages. Each experimental
point was performed 5-7 times using independent aortae. Significance
was evaluated by standard t test analysis prior to
conversion to percentages and within each experimental group.
Flow Cytometry--
Quiescent HDECs were plated in phenol
red-free EBM supplemented with 0.1% charcoal-filtered serum, 50 ng/ml
VEGF, and 2 ng/ml FGF-2. Cells were treated with 1 µM
progesterone, 10 µM R5020, or vehicle. At the indicated
time points, cells were removed by incubation with trypsin, fixed with
70% ethanol, and incubated with RNase A (10 µg/ml). Staining of DNA
was accomplished by incubation with 50 µg/ml propidium iodide (Sigma)
containing 1 µg/ml RNase A. Dead cells were excluded from analysis by
gating in FL3. DNA fluorescence of nuclei was measured with a FACScan
flow cytometer (Becton Dickinson, Bedford, MA), and percentages of
cells in G0 and G1, S and G2, and M
phases of cell cycle were analyzed using FACScan software programs.
Western Blot Analysis--
An equal number of quiescent HDECs
were passed to 6-well plates in phenol red-free EBM supplemented with
1% FCS and 50 ng/ml VEGF, in the presence of either progesterone,
R5020, or vehicle control. At the times indicated, monolayers were
processed, separated by SDS-polyacrylamide gel electrophoresis, and
transferred to nitrocellulose membranes (Schleicher & Schuell). After
incubation with primary antibodies, immune complexes were visualized by
ECL (Amersham Pharmacia Biotech). Antibodies included cyclin A (BF683) and cyclin E (HE12) from Santa Cruz Biotechnology Inc. (Santa Cruz,
CA), and pRb (G3-245) from Pharmingen.
Statistics--
Statistical analysis were done using In-Stat
software (Graph Pad Software) for Macintosh. All categorical data
(scanning densitometry) is presented as mean ± S.D. when repeated
measures were done. Assuming normal distributions, data were analyzed
by one-way analysis of variance, followed by either the t
test with Dunnett test for comparisons between groups or the
Student-Newman-Kleus test for multiple comparisons between groups.
Progesterone Receptor Is Expressed by Several Types of Endothelial
Cells--
Presence of progesterone receptor was evaluated by three
independent assays: (a) immunocytochemistry on tissue
sections, (b) Northern blots of purified endothelial cell
cultures, and (c) immunoprecipitation of cell culture
extracts. Immunohistochemical analysis with an PR-specific monoclonal
antibody revealed expression of PR in the endothelium of normal (Fig.
1A, a-c) and atherosclerotic (Fig. 1A, d-f) arteries. Double immunolabeling with von
Willebrand's factor was performed to verify the endothelial nature of
these cells. Expression of PR in the endothelium was patchy, with about 25-30% of the total number of endothelial cells lining the lumen positive for PR (Fig. 1A). At present, it is not clear
whether this indicates endothelial heterogeneity or a particular
physiological state. Nevertheless, the proportion of PR-positive cells
in the endothelium of vessels varied in different organs, with cycling endometrium showing the higher proportion of endothelial PR-positive cells (25). As described previously, we also observed PR expression in
the smooth muscle layer (8). In our preparations, the expression was
scant and distributed center and lower half of the tunica media, an
area not seen in Fig. 1A due to the magnification.
The relative levels of PR mRNA were assessed by Northern analysis
(Fig. 1B). Several purified endothelial cell cultures
isolated from skin, lung, endometrium, and coronary vessels expressed
PR mRNA. However, the relative levels of transcript varied
significantly among the cell types examined. In addition, whereas
expression of the receptor was maintained in vitro in most
endothelial cells tested, levels decreased with increase passage
number. The presence of PR protein was evaluated by immunoprecipitation
of cell extracts (Fig. 1C). The assays were performed with
the same antibody used on immunocytochemistry. Both forms of the PR
receptor, PR-B (120 kDa) and PR-A (94 kDa) were detected at a similar
ratio. The relative levels of PR on endothelial cells, however, were
about
Further characterization of PR on endothelial cells was provided by
direct binding assays (Fig.
2A). Cell extracts of
endothelial cell cultures were subjected to incubation with
[3H]R5020, a progestin agonist. Scatchard analysis showed
a unique high affinity binding protein with Kd = 6.1 × 10
Functional evaluation of PR activity was determined by transactivation
assays (Fig. 2B). Presence of intracellular progestins lead
to dimerization of PR and binding to specific cis-acting sequences (PREs). We transfected several endothelial cell types with a
construct containing 6 tandem PRE repeats linked to the luciferase
gene. In the absence of progestins, there was little to no detectable
luciferase; however, treatment of transfected cultures with progestins
induced transactivation and expression of luciferase. These experiments
were confirmed with at least three independent strains of each cell
type. Interestingly, we detected consistent trends in the
transactivation assay. Endothelial cells derived from lung and
umbilical cord gave the lowest luciferase levels, and coronary and
dermal endothelial cells showed the highest. In these assays, low
passage number was an essential requirement for a positive
transactivation response and correlated with higher expression of PR.
To determine the effect of progestins on endothelial cell function, we
evaluated attachment, spreading, migration, invasion, and proliferation
of endothelial cells in the presence of progestins. Although a clear
effect was detected on proliferation assays, no significant alterations
were seen in any of the other endothelial cell functions tested (data
not shown).
Progesterone Is an Effective Suppressor of Endothelial Cell
Proliferation--
Inhibition of [3H]thymidine
incorporation by progesterone was dose-dependent (Fig.
3A). Significant growth
suppression was seen with levels as low as 10 nM. At 100 nM, progesterone inhibited endothelial cell proliferation
by 30-35% and up to 45-50% at 1 µM after 48 h of
treatment. This effect was confirmed with several independent strains
of human dermal endothelial cells (17 strains), human coronary
endothelial cells (3 strains), bovine aortic endothelial cells (5 strains), and human endometrial endothelial cells (9 strains). The
reduction of [3H]thymidine incorporation mediated by
progesterone reflected changes in cell number, as confirmed in parallel
experiments for which cell counts were performed at the end of 4 days
of treatment (Fig. 3B). Cells incubated with progestins were
not permanently suppressed, because withdrawal of progestins enabled
endothelial cultures to respond to mitogens at equal intensity as
controls.
The specificity of the response mediated by progesterone was further
evaluated with the use of R5020, a progesterone agonist more stable
than progesterone. Synchronized endothelial cultures treated with R5020
showed inhibition that was sustained through the entire cell cycle.
Fig. 3C shows the effect of R5020 on cell cycle, as
determined by thymidine incorporation. A significant reduction
(72-75%) in thymidine incorporation was seen at the peak in S phase
(48 h). It should be noted that this effect is cumulative over time, an
observation that is consistent with cell cycle arrest. A relatively
short but reproducible delay of 8-12 h was also noted during the peak
of S phase on treated cultures.
To determine whether reduction in total number of cells was due to
toxicity, apoptosis, or cell cycle arrest, we counted the number of
cells in several time points during the entire assay (48 h), finding no
change with progesterone treatment prior to or during S phase.
Furthermore, progesterone did not promote changes in cell shape or
spreading, nor did it induce apoptotic bodies, as evaluated by
microscopic inspection of the cultures. The data are consistent with a
specific cell cycle arrest rather than cell death.
The Effect of Progesterone on Cell Cycle Arrest Is
PR-mediated--
Two experiments added to the specificity of the
effect mediated by progesterone and provided support that the
suppression on cell cycle required and was dependent on the presence of
PR. Fig. 4A shows that the
effect of progesterone was blocked by preincubation of endothelial
cultures with increasing concentrations of RU486, a potent antagonist
of progesterone that specifically binds to PR (29, 30). Similar
levels of RU486 alone had no significant effect on thymidine
incorporation.
The role of PR on the progesterone-mediated inhibition was further
confirmed by experiments in which endothelial cells derived from PRKO
mice were treated with the hormone (5). Proliferation of endothelial
cells isolated from wild-type animals were inhibited by both
progesterone and R5020 at kinetics similar than those described for
HDECs. In contrast, endothelial cells from PRKO mice were not affected
by the same treatment (Fig. 4B). These findings also
confirmed the specificity and lack of toxicity of progesterone on
endothelial cells and demonstrate the requirement of PR for cell cycle arrest.
The inhibitory effect of progestins was applicable to a variety of
mitogenic signals, including basic fibroblast growth factor, vascular
permeability/endothelial growth factor, and fetal calf serum, alone or
in combination. The relative levels of inhibition were equivalent with
each of these factors. These results indicated that the effect mediated
by progesterone was mostly downstream from these effectors. The
biological significance of our findings was tested in ex
vivo models of vascular injury.
Progesterone Affects Re-endothelialization of Injured
Aortae--
The role of progesterone on inhibition of endothelial cell
growth could have relevant implications in the regulation of
angiogenesis and vascular repair. To evaluate the possible role of
progestins in the repair of endothelial denuded areas, we performed
intima injury experiments in aortae of wild-type and PRKO mice, the
latter as experimental controls. Following dissection of aortae, a
2-4-mm endothelial area was removed. The extension of the injury was assessed by staining with Evans blue and recorded at time 0. Exposed aortae were treated in vitro with endothelial growth factors
in the presence or absence of R5020 or progesterone for 4 consecutive days and were then restained with Evans blue to evaluate endothelial regrowth (Fig. 5A). Evans blue
is a vital dye that binds to albumin and to other proteins in the
extracellular matrix (31). Intact endothelium is impermeable to this
dye, remaining white (unstained), whereas areas that lack endothelium
stain in blue. This method has been extensively used to reveal lumenal
areas that expose the smooth muscle layer of the media as a consequence
of endothelial injury (32-34). In our assays, the initial extent of
blue staining was compared with the same restained specimen after
treatment as indicated. Only the regrowth of the endothelial layer
blocked binding of the dye. Significant re-endothelialization was
evident in the absence of progestins in both wild-type and PRKO mice. However, presence of progestins greatly suppressed (77%) endothelial repair in aortae from wild-type animals (Fig. 5B). No
difference from controls were observed in the rate of endothelial
repair in PRKO aortae treated with progesterone. The experiments were performed in 10 wild-type and 12 PRKO mice and included both males and
females. These data provided physiological relevance to our findings
and confirmed the suppressive effects of progestins on the
endothelium.
To further understand the mechanism of action of progestins on
endothelial proliferation, we performed a series of cell cycle analysis
in the presence and absence of the hormone.
Progesterone Arrests Endothelial Cells in G1--
FACS
analysis of endothelial cultures treated with progesterone or R5020
showed that these compounds promoted cell cycle arrest in
G0/G1 (Fig.
6A). The arrest was cumulative
at the expense of cells in S, G2, and M phases. Retention
of cells in G1 increased over time, with 38% of the cells
at 24 h, 47% at 48 h, and near 80% at 72 h. These
experiments also confirmed absence of apoptosis upon R5020 treatment.
In control cultures (cells seeded on bovine serum albumin-coated plates
(50 µg/ml)), apoptosis was revealed as a peak of cells with DNA
content lower than G0/G1. In contrast, cultures
treated with R5020, progesterone, or vehicle did not show such a
population of cells.
Cell cycle progression from G0/G1 to S is
controlled by changes in activity of G1 phase
cyclin-dependent kinases (Cdks) that regulate the
phosphorylation state of pRb, a key protein for entry in S phase (35).
We analyzed pRb phosphorylation to determine whether changes in Cdk
activity might result from progesterone treatment (Fig. 6B).
Untreated cells showed maximum levels of pRb phosphorylation by 24 h, with decreased phosphorylation thereafter. In contrast, cultures
exposed to R5020 showed a 12-24-h delay in pRb phosphorylation when
compared with controls. These results are in agreement with the
reduction in the S phase fraction seen by FACS analysis and thymidine
incorporation. Interestingly, we have observed a hypophosphorylated
form of pRb upon R5020 treatment (doublet in Fig. 6B, R5020,
4-24 h). At least three Cdk complexes mediate pRb phosphorylation,
specifically Cdk2-cyclin A, Cdk2-cyclin E, and Cdk4-cyclin D1 (36).
These pRb inactivating kinases, however, phosphorylate pRb differently,
providing several electrophoretic mobility forms of the protein (36).
It appears that treatment with R5020 alters the profile of pRb
phosphorylation in addition of delaying hyperphosphorylation.
Also, these alterations might relate to the direct effect of
progesterone on cyclins expression, as mentioned below.
To further support the effects of progesterone on cell cycle
progression, we examined the relative protein levels of cyclins E and
A. Expression of these proteins is associated with checkpoints and
progression through early and late G1, respectively. The
pattern of expression for cyclin E and A is altered upon treatment with R5020 (Fig. 6C). Whereas vehicle-treated cells showed a peak
of cyclin E and cyclin A expression at 24-36 h and 36-48 h,
respectively, the levels of these proteins on R5020 treated cells was
sustained as long as 60 h for both. This pattern reflects a
temporal extension of G1 phase. The experiments did not
distinguish whether decrease in protein levels of cyclins E and A are a
cause or a consequence of pRb regulation, because these cyclins have
been shown to act both upstream and downstream of pRb. Nevertheless,
the results demonstrate that progesterone affects expression of several
proteins involved in cell cycle progression, and as cause or
consequence, Cdk activity is also affected, resulting in inhibition and
delay of pRb phosphorylation. We also evaluated p27 and p21 levels upon treatment with progesterone, and no significant alteration was detected
in either of these proteins.
Further analysis is required to clarify the specific mechanism by which
progestins mediate endothelial cell cycle arrest. However, this
discrete analysis provided support to our findings and indicated that
the target of progestins is likely upstream pRb phosphorylation during
G1.
In this study, we have provided functional support to the presence
of progesterone receptor on vascular endothelium. Although expression
of progesterone receptor has been previously reported in the intima of
blood vessels using biochemical and immunocytochemical approaches (19,
20), to our knowledge, this is the first study that performs a
broad-based analysis on several endothelial types and that provides a
functional relevance to this receptor on endothelial biology.
The use of steroid hormones in postmenopausal replacement therapy has
been associated with prevention of cardiovascular disease. However, the
contribution of progesterone to the replacement regime has been
controversial. Although experimental studies evaluating intimal
thickness (37) and bromodeoxyuridine incorporation (38) conclude that
progesterone directly inhibits the atheroprotective effect of estrogen,
several epidemiological and in vivo studies demonstrate
otherwise (39, 40). The finding that progesterone inhibits
re-endothelialization of denuded aortae suggests that this hormone
could have an opposite effect on estradiol in endothelial repair of
denuded atherosclerotic lesions (41).
Atherosclerosis is a multifactorial process and the principal
contributor to myocardial and cerebral infarction (42). Response to
injury is one of the favored hypotheses for development of atherogenesis. It postulates that an alteration of the intima by
various risk factors (such as mechanical injury, chemically altered low
density lipoprotein, viruses, or toxins) initiates a primary
endothelial cell dysfunction that leads to subsequent vascular changes,
giving rise to the initial atherosclerotic lesion. The plaque
progresses by the accumulation of layers of smooth muscle, macrophages,
and foam cells; further deposition of extracellular matrix; and
possibly neovascularization. Progression or rupture of the plaque is
frequently associated with physical disruption of the endothelium.
Because of the role of the endothelium in providing an antithrombotic
and anticoagulant surface, rapid endothelial repair is of importance to
the containment of atherosclerotic lesions. Although progesterone has
shown suppressive effects on smooth muscle cell proliferation in
vitro (8), its participation on intimal endothelial repair,
according to this study, appears to be equally inhibitory and, by
extension, possibly deleterious to the endothelial healing of an
exposed lesion.
The inhibitory effect of progesterone on endothelial proliferation also
provides an explanation for the episodes of vessel breakdown and
irregular bleeding associated with progestin-based contraceptives (21).
Physiological, but constant, levels of circulating progestins most
likely affect the high mitotic rate associated with the endometrial
vasculature,3 leading to
capillary rupture. Furthermore, progesterone has been implicated in the
suppression of tumor-induced neovascularization (43).
In this study, we showed that progesterone partially blocks and delays
endothelial cell cycle progression through a receptor-mediated mechanism that involves changes in expression of cell cycle proteins, cyclins E and A, and changes in pRb phosphorylation. The participation of PR in the regulation of cell cycle has been controversial, with
studies indicating that progesterone stimulates, inhibits, or does not
alter cell cycle progression even on the same cell type (8, 44-46).
When inhibitory in mammary epithelial cells, the mechanism by which
progesterone mediates cell cycle arrest has been shown to implicate
both suppression of Cdk activity (47) and up-regulation of p21 (48). In
endothelial cells, however, it is likely that progesterone acts by
regulation of Cdk activity only, because we have not detected changes
in either p21 nor p27 expression levels (data not shown). It has now
become increasingly clear that the functional contribution of PR as a
transcription factor is also dependent on the expression of several
binding proteins that modulate PR activity (49-52). Whether or not
these binding proteins play a role in the regulation of endothelial cell cycle or any other cell type is, at this point, not clear and
deserves further investigation. Nevertheless, we found that endothelial
cell proliferation was suppressed by physiological levels of
17- In conclusion, the presence of progesterone receptor in endothelial
cells and the direct effect of progesterone described here can be of
physiological relevance to the balance between potentiation of
endothelial growth, mediated by estradiol, and suppressive signals,
mediated by progesterone. This balance could play an important role in
the regulation of angiogenesis in the endometrium and corpus luteus
during the menstrual cycle. Nonetheless, constant levels of progestins
in HRT could be counter-productive. Although the benefits of
progestins, as first or second line therapy in the treatment of breast
and endometrial carcinoma, have been widely acknowledged, the long-term
use of progestins as contraceptives or for the prophylaxis of menopause
should be under closer scrutiny. It is clear from these and other data
that progestins may have direct growth-inhibitory actions in
nonreproductive sites, apart from their ability to inhibit
estrogen-mediated cell proliferation in such classical reproductive
targets as the endometrium.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-methyl-3H]R5020 (NEN Life Science
Products). Incubation was allowed to take place in the presence or
absence of 100-fold molar excess of R5020 or estradiol (as a negative
control competitor). Binding was assessed by liquid scintillation, and
Scatchard plots were used to evaluate the data.
RESULTS
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Fig. 1.
Progesterone receptor expression in
endothelial cells. A, immunolocalization of PR in the
nuclei of endothelial cells. Human specimens from normal
(a-c) and atherosclerotic (d-f) vessels were
incubated simultaneously with antibodies to PR (1298) and vWF. PR was
identified with a Texas Red-conjugated secondary antibody (b
and e), and vWF was detected with a fluorescein
isothiocyanate-conjugated antibody (a and d). The
sections were counterstained with Hoechst to visualize nuclei
(c and f). Analysis of the same area was done
individually in three excitation channels using a Zeiss Axiophot.
White arrows indicate positive nuclei, and red
arrows indicate negative nuclei. Note the expansion of the
neointima (N) in the atherosclerotic lesion (d,
bracket). B, Northern blot analysis for PR expression.
Shown are the results for stromal cells, dermal epithelial cells, human
lung endothelial cells (HLEC), human endometrial endothelial
cells (HEEC), human dermal endothelial cells
(HDEC), and human coronary endothelial cells
(HCEC). C, immunoblot analysis for PR expression.
5 µg of T47D protein extract and 200 µg of different HDEC strains
(passages 3 and 4) were immunoprecipitated with anti-PR (1298) and
further identified on Western blots with the same anti-PR. Both forms
of PR, PR-A and PR-B, were detected and are indicated by
arrows.
of the levels of PR on T47D.
9 M, which could be competed
with cold R5020 but was unaffected by 17
-estradiol. In addition,
radioligand binding assays revealed that early passage endothelial
cultures (HDECs) had as many as 105 receptors/cell, but a
reduction of up to 100-fold was seen after three passages in
vitro.
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Fig. 2.
Progesterone promotes binding and induces
transactivation on endothelial cells. A, Scatchard
analysis of progesterone receptors on HDECs. Cultures of HDECs (passage
3) were subjected to cytosolic extraction, and aliquots were incubated
with increasing concentrations (0.2-30 nM) of
[3H]R5020. Scatchard plots were used to analyze the data.
B, transactivation of PR cis-acting element by
progesterone on endothelial cells. Cultures of endometrial stromal and
different types of endothelial cells were transfected in triplicate
with control plasmid (pCMV) or a plasmid containing 6 tandem repeats of
the progesterone responsive element (pCMV-PRE) linked to the luciferase
reporter gene. 24 h after transfection, cultures were treated
either with vehicle or progesterone (0.5 µM) for 6 h. Luciferase activity was assessed, normalized to total protein, and
expressed in the histogram. HEEC, human endometrial
endothelial cells; HUVEC, human umbilical vein endothelial
cells; HLEC, human lung endothelial cells; HCEC,
human coronary endothelial cells; BAEC, bovine aortic
endothelial cells. Columns represent average of three
independent assays (± S.D.).
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Fig. 3.
Effect of progesterone on endothelial cell
proliferation. A, HDECs and human coronary endothelial
cells (HCEC) were synchronized and plated on 24-well plates
in the defined media with vehicle or increasing amounts of
progesterone. Cells were grown for 48 h, and
[3H]thymidine was added during the last 12 h. Assays
were performed in quadruplicate. B, equal numbers of
quiescent HDECs were plated on 12-well plates in EBM supplemented with
0.05% charcoal-filtered serum, in the presence or absence of FGF-2 (2 ng/ml) and varied concentrations of R5020. The assay was allowed to
continue for 4 days. Cells were trypsinized and counted. Assays were
done in triplicate. C, quiescent HDECs were plated on
24-well plates in EBM supplemented with 0.1% FCS and VEGF (50 ng/ml)
in the presence or absence of R5020 (1 µM). A pulse of
[3H]thymidine was added 8 h before for each time
point, and cells were harvested every 8 h for a total of 120 h. Bars in all panels indicate S.E.
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Fig. 4.
Inhibition of endothelial cell proliferation
is mediated by PR. A, HDECs were plated on 24-well
plates in EBM containing 0.1% charcoal-filtered serum and 2 ng/ml
FGF-2. Treatments as indicated in the figure included progesterone (1 µM) and RU486 (0.1, 0.5, and 1 µM).
Incubations were performed for 48 h with an 8-h pulse of
[3H]thymidine at the end of the treatment. Each assay was
done in triplicate. B, brain endothelial cells were isolated
from wild-type and PRKO mice and cultured in EBM containing 0.1% FCS.
Cells were then treated with progesterone (5 or 0.5 µM)
or R5020 (5 µM). Cells were allowed to grow for 2 days,
and a pulse of [3H]thymidine was added during the last
8 h. Bars in all panels indicate S.E.
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Fig. 5.
Effect of progesterone on
re-endothelialization of injured aortae. A, aortae from
wild-type (a-d) and PRKO (e-h) mice were
dissected, and a 2-4-mm endothelial wound was inflicted as described
under "Experimental Procedures." Staining with Evans blue revealed
the area of denuded endothelium at day 0 (a, c, e, and
g). Specimens were then cultured in EBM supplemented with
1% FCS and 2 ng/ml FGF-2 in the presence (c, d, g, and
h) or absence (a, b, e, and f) of 1 µM progesterone during 4 days at 37 °C. At the end of
the incubation time, aortae were restained with Evans blue (b, d,
f, and h). Photomicrographs were taken at days 0 and 4. B, evaluation of re-endothelialization in aortae of
wild-type and PRKO mice was performed by direct measurement of the
injured area in the midsection of the aortae (arrows in
panel A) at days 0 and 4. Reduction of the injured area in
control was considered 100%. A total of six independent experiments
were performed. Statistical analysis of the control and progesterone
subgroups in wild-type mice was statistically significant
(p < 0.001).
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Fig. 6.
Progesterone mediates cell cycle arrests of
endothelial cells in G1. A, FACS analysis.
Quiescent HDECs were plated onto Vitrogen-coated 100-mm2
dishes in EBM containing 50 ng/ml VEGF, 2 ng/ml FGF-2, and 0.1%
charcoal-filtered serum in the presence or absence of progesterone (1 µM) for 72 h. Cells were harvested and processed for
cell cycle analysis (see under "Experimental Procedures").
Distribution of cells within each phase of the cycle corresponds to
average of three independent plates. B, effect of R5020 on
pRb phosphorylation. Quiescent HDECs were treated with R5020 (1 µM) or vehicle and were harvested at the indicated times.
Cell lysates were resolved on 7% SDS-polyacrylamide gel
electrophoresis gel and immunoblotted with a pRb antibody. The
top band corresponds to the hyperphosphorylated form
(ppRb); the bottom band indicates the
hypophosphorylated form (pRb). C, effect of R5020
on expression of cyclin E and cyclin A. Quiescent HDECs were treated
with R5020 (1 µM) or vehicle and were harvested at the
indicated times. Cell lysates were resolved on 12.5%
SDS-polyacrylamide gel electrophoresis gels and immunoblotted with
antibodies for cyclin E or cyclin A.
DISCUSSION
-hydroxy-progesterone in every endothelial cell type examined.
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ACKNOWLEDGEMENTS |
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We thank Katie Davies for excellent technical help on the isolation of endothelial cells and Dr. Tim Lane for discussions and comments during the progression of this project.
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FOOTNOTES |
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* This work was supported by National Institutes of Health, NCI Grants RO3CA70559-01 and R29CA63356-01 (to M. L. I.-A.) and a fellowship from Caixa Balears Sa Nostra and Real Colegio Complutense (Spain) (to F. V.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors made equivalent contributions.
** To whom correspondence should be addressed: Dept. of Molecular Cell and Developmental Biology, UCLA, Molecular Biology Institute, R559, 611 Circle Dr. East, Los Angeles, CA 90095-1570. Tel.: 310-794-5763; Fax: 310-794-5766; E-mail: arispe{at}mbi.ucla.edu.
The abbreviations used are: PR, progesterone receptor; PRKO, PR knockout; FCS, fetal calf serum; FGF-2, basic fibroblast growth factor; HDEC, human dermal endothelial cell; HRT, hormone replacement therapy; VEGF, vascular endothelial growth factor; pRb, retinoblastoma protein; Cdk, cyclin-dependent kinase; FACS, fluorescence-activated cell sorter; PRE, progesterone-responsive element.
2 D. Edwards, unpublished data.
3 M. Graubert, M. Lombardo, M. A. Ortega, L. F. Brown, B. Kessel, J. F. Mortola, and M. L. Iruela-Arispe, unpublished observations.
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REFERENCES |
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