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Regular Articles |
From the Department of Obstetrics and Gynecology,*
Division of Reproductive Endocrinology, Beth Israel Deaconess Medical
Center, Harvard Medical School, Boston, Massachusetts; and the
Department of Molecular, Cell, and Developmental Biology and the
Molecular Biology Institute,
University of
California–Los Angeles, Los Angeles, California
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, and
interleukin-1ß. Both VEGF receptors, FLT-1 and KDR,
followed a similar pattern. However, functional activity of
KDR, as determined by phosphorylation studies, revealed
activation in the late menstrual and early proliferative phases. The
degree of KDR phosphorylation was inversely correlated with the
presence of sFLT-1. Endothelial cell proliferation analysis in
endometrium showed a peak during the late menstrual and early
proliferative phases in concert with the presence of VEGF, VEGF
receptor phosphorylation, and decrease of sFLT-1.
Together, these results suggest that VEGF receptor
activation and the subsequent modulation of sFLT-1 in the late
menstrual phase likely contributes to the onset of angiogenesis and
endothelial repair in the human endometrium.
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Introduction |
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It is evident that a large number of angiogenic growth factors might contribute to the initiation, progression, and morphogenesis of blood vessels associated with endometrial repair. Several angiogenic cytokines have been identified in the human endometrium including basic fibroblast growth factor-2, platelet-derived growth factor, epidermal growth factor, and transforming growth factor-ß (TGF-ß), although vascular permeability/vascular endothelial growth factor seems to be a major candidate for modulating the angiogenic response.4,5,10,11
The role of VEGF as a mediator of angiogenesis during menstrual repair has been intensively investigated during the last decade. Most of the studies, however, have been descriptive in nature, providing either immunocytochemistry, in situ hybridization, or reverse transcriptase-polymerization chain reaction (RT-PCR) analysis for assessment of VEGF levels. This can be attributed, for the most part, to the tremendous limitations imposed by the uniqueness of the human endometrial cycle that cannot be reproduced in most animal models, except for a subset of primates. Functional studies addressing the relevance of the VEGF-signaling system to the female reproductive tract have been done in rodent models. Treatment of female rats with a truncated soluble form of the FLT-1 receptor resulted in virtually complete suppression of angiogenesis in the corpus luteum with associated maturation failure of the endometrium.12 The truncated Flt-1 molecule acted in a dominant-negative manner, diminishing association of VEGF to its transmembrane receptors and impacting subsequent signaling pathways.12 This elegant and important manuscript emphasized the relevance of the VEGF-signaling to the maintenance and normal physiology of the ovary and endometrium. However, these studies could not shed light on the participation of this signaling pathway in the human endometrial cycling.
In humans, VEGF mRNA is present throughout the endometrial cycle and seems to be increased in the secretory phase,11,13 but this expression pattern does not parallel the predicted temporal stage associated with neovascularization and vascular repair that follows menstruation.14 Serum levels of VEGF from woman at all stages of the endometrial cycle have been the focus of intense investigation.15 Nonetheless, no significant changes and a complete lack of cyclicity was consistently found (M. L. Iruela-Arispe, unpublished results).15 Interestingly, patients subjected to in vitro fertilization treatment, which involves significant doses of hormones, showed elevated levels of VEGF in serum during the luteal phase of the cycle.15 This suggests that, at least in part, endometrial hormones contribute to VEGF levels. To support this concept, hormonal-based contraceptives have been shown to have an effect in the distribution of VEGF from the stroma to the glandular compartment16 and also seem to predispose the endometrium to an increased vascular fragility.17,18 Nonetheless, the relative contribution of VEGF to the normal physiology of the endometrial cycle remains elusive and additional investigations are required to: 1) concretely understand the contribution of steroid hormones in VEGF expression during normal physiology in concert with other endometrial confounding factors, in particular hypoxia; and 2) gain further insight as to the orchestrated regulation of VEGF, VEGF receptor expression, and signaling on endothelial cells during the endometrial cycle.
The present study was aimed at elucidating the role of the VEGF and VEGF receptors in modulating the initial wave of angiogenesis associated with postmenstrual repair. Using endometrial biopsy specimens from a cohort of normal ovulating women, our findings revealed a sophisticated regulatory control of VEGF-receptor activation that coincides with the onset of endothelial proliferation and vascular reconstitution of the tissue.
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Materials and Methods |
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Entry criteria for this study included healthy, nonsmoking women aged 18 to 36 years, with regular menses, normal cervical cytology, no history of ovulatory dysfunction, endometriosis, infertility, or eating disorders. The study participants had no history of Norplant or Depo-Provera use in the last year, no oral contraceptive use in the past 3 months, and were more than 6 months postpartum and nonlactating. After receiving informed consent, patients meeting entry criteria underwent a physical examination, blood draw, urine pregnancy test, and endometrial aspiration biopsy using a Pipelle instrument (Cooper Surgical, Shelton, CT). The protocol was approved by the Committee on Clinical Investigations, Beth Israel Deaconess Medical Center. Endometrial tissue was placed in modified Eagle’s media and immediately processed for RNA extraction, cell isolation, and histology. Study participants’ serum was pooled and stored at -70°C until completion of recruitment.
Study Groups and Serum Hormone Assay
The study participants’ serum was measured for estradiol, progesterone, and luteinizing hormone in duplicate aliquots using an AutoDELPHIA immunoassay (Wallac, Turku, Finland) (courtesy of Dr. James Faix, Beth Israel Deaconess Core RIA Laboratory). The study participants were divided into study groups based on cycle day of biopsy, hormone levels, and endometrial dating.19 The late proliferative phase group was assessed by serology as having pre-ovulatory progesterone levels (<7 nmol/L), and significantly higher estradiol and luteinizing hormone levels than the early proliferative group. Study participants in the secretory groups were considered when serum progesterone levels were indicative of ovulation (>7 nmol/L). Sixty-nine study participants entered the study, and eight could not have endometrial biopsy completed because of discomfort with procedure. Sixty-one endometrial biopsies were obtained for study: one late proliferative phase sample was excluded for a progesterone level suggestive of premature luteinization (>7 nmol/L), and one sample was excluded because of error in processing, leaving 59 biopsies available for study.
Materials
Paraformaldehyde, Ficoll-400, polyvinylpyrrolidone, salmon
testicular DNA, dextran sulfate, RNase A, progesterone,
17-ß-estradiol, TGF-, and interleukin (IL)-1ß were purchased
from Sigma Chemical Co. (St. Louis, MO).
Trypsin-ethylenediaminetetraacetic acid, collagenase, phenol-free
medium, and antibiotics were purchased from Life Technologies, Inc.
(Gaithersburg, MD). Charcoal-filtered fetal calf serum was supplied
from Cocalico Biologicals (Reamstown, PA). The random-primed
DNA-labeling kit was purchased from Amersham Corp. (Arlington Heights,
IL). [32P]-dCTP was purchased from DuPont NEN
(Boston, MA). Nylon membranes (Nytran) were purchased from Schleicher
and Schuell (Keene, NH). All other reagents used in this study were of
the highest purity available from Fisher Biochemicals (Pittsburgh, PA).
Northern Analysis
Total RNA was purified from either minced whole tissue or purified
cells by guanidinum-isothiocyanate extraction.20
Poly(A+)
enriched RNA was isolated using an oligo(dT)-streptavidin magnetic bead
separation kit from Boehringer Mannheim (Indianapolis, IN). Samples
were subjected to electrophoresis on a denaturing 1% agarose gel and
transferred to nylon membranes (Nytran). Northern blots were hybridized
with [32P]-labeled cDNA probes. The following
cDNA fragments were used in this study: 1) a 930-bp EcoRI
fragment that recognizes all VEGF isoforms (a generous gift from N.
Ferrara, Genentech); 2) a 1-kb EcoRI/XhoI
fragments of VEGF-B and VEGF-C cDNAs (obtained by RT-PCR); 3) a 634-bp
probe, generated from the HindIII-BamHI
restriction fragment (bp1661 to 2294) of sFLT-1 (obtained by RT-PCR);
4) a KDR cDNA fragment that corresponds to a 300-bp
EcoRI/BamHI fragment subcloned into pGEM3 (a gift
from Kevin Claffey; University of Connecticut); 5) a 925-bp
EcoRI TGF- cDNA fragment (American Type Culture
Collection, Rockville, MD); 6) a 1-kb SmaI/BamHI
fragment for IL-1ß; and 7) a 2.47-kb BamHI GLUT-1 cDNA
fragment (a generous gift from Dr. Kevin Claffey, University of
Connecticut). Membranes were exposed to Biomax MS film (Kodak,
Rochester, NY), and densitometric analysis of autoradiograms were
performed by scanning densitometry using a Vista S-12 scanner (UMAX,
Taiwan, R.O.C.), and Molecular Analyst software (Bio-Rad, Hercules, CA)
for Power Macintosh (Apple, Cupertino, CA). All densitometric data were
normalized for loading and transfer efficiency to expression of 36B4 (a
ribosome-associated protein).21
Isolation of Human Endometrial Stromal Cells
We followed previously described procedures with few modifications.21 Specifically, endometrial samples were minced into 1- to 2-mm3 pieces under sterile conditions in a laminar flow hood and digested with collagenase at 37°C for 2 hours. After digestion, the cell suspension was filtered through a 70-µm nylon mesh to remove undigested fragments. Cells were spun and washed four times in Dulbecco’s modified Eagle’s medium containing a fivefold excess of antibiotics (penicillin, streptomycin, and gentamicin). Isolated cells were plated on tissue culture dishes previously coated with 50 µg/ml of vitrogen (Collagen Biomaterials, Palo Alto, CA). Cells were allowed to attach for 30 minutes; nonattached cells were removed by aspiration. Stromal cultures were evaluated for expression of estrogen receptor and progesterone receptor. Functional characterization of estrogen receptor and progesterone receptor was performed by transactivation of luciferase after steroid treatment, as previously performed.22 Briefly, transfection of stromal cultures was performed with 1) a luciferase reporter construct containing six-tandem repeats of the estrogen receptor-binding motif (a generous gift from Dr. Miller, Dana Farber Cancer Institute, Boston, MA), or 2) a luciferase reporter construct driven by four-tandem repeats of the progesterone receptor CIS acting element (a generous gift from Dr. Sam Lee, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA), and 3) a plasmid containing CMV renilla to control for transfection efficiency. Lipofectamine (Life Technologies, Inc.) was used in all transfections following the manufacturer’s recommendations. Assays were performed in triplicate and were normalized for both transfection efficiency [with a renilla construct (Promega)] and total protein. Expression of luciferase and renilla was evaluated with a luminometer (Wallac, Gaithersburg, MD). Stromal cells were no longer used when functional levels of one or both receptors dropped 30% below the levels established for that culture at passage 2 (the decrease in receptor was seen in some cultures as early as passage 6 and as late as passage 10 in other cultures).
Treatment of stromal cultures with estradiol (Sigma), progesterone
(Sigma), recombinant TGF-, and IL-1ß (Life Technologies, Inc.) was
performed at the times and concentrations indicated in the figure
legends. All cultures were incubated in serum-free and phenol-red-free
media for 16 hours before treatment to remove potential
confounding factors that could interact with steroid receptors.
For neutralization experiments, antibodies against human TGF-
(polyclonal made in rabbit) and IL-1ß (monoclonal made in mouse) were
purchased from Leinco Technologies (St. Louis, MO). Antibodies were
incubated with the related growth factor for 3 hours before cell
treatment (10 ng of growth factor, 30 µg of antibody). Controls
included treatment of cultures with antibodies alone and incubation of
the growth factors with rabbit (for TGF-) or mouse (for IL-1ß)
IgG. Treatment of cells followed for 4 hours and evaluation was
performed by Northern analysis.
Hypoxia experiments were done using a Heraeus incubator using 3%
O2 and 5% CO2 gas
concentrations. We have selected this range of oxygen tension, as it
has been shown that transcriptional response to hypoxia (particularly
through induction of HIF-1) is triggered at oxygen concentrations
ranging from 3 to 5%.23
Enzyme-Linked Immunosorbent Assays (ELISAs) for Detection of VEGF
VEGF protein was evaluated on sandwich ELISAs. Capture of VEGF was
accomplished with a bound chicken IgY antibody to human VEGF (a gift
from Dr. Don Senger, Beth Israel Deaconess Medical Center, Boston, MA).
Followed by a mouse -VEGF antibody (also from Don Senger). A
peroxidase-conjugated goat anti-mouse IgG was used as secondary
antibody (KPL, Gaithersburg, MD). Detection was aided by using KPL
enhancer substrate, reactions were measured on a luminometer. Values
were compared to a VEGF curve performed in parallel.
Immunoprecipitation and Western Blot Analysis
For immunoprecipitation of VEGF, tissue samples were solubilized in extraction buffer (50 mmol/L Tris, pH 7.5, 2% glycerol, 150 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid, 1% Triton X-100) containing proteinase inhibitors (2 mmol/L phenylmethylsulfonyl fluoride, 10 mmol/L NaF, 1 mmol/L NaVO4, and 10 µg/ml of each aprotinin, leupeptin, and pepstatin A). After centrifugation at 10,000 rpm for 30 minutes, protein extracts were mixed with a slurry of heparin-Sepharose CL-6B (Pharmacia) and incubated overnight rocking at 4°C. Beads were harvested by centrifugation, washed with 450 mmol/L NaCl, and eluted with 1.5 mol/L NaCl. Protein was dialyzed against phosphate-buffered saline (PBS) and quantified using the Bio-Rad protein assay system (Bio-Rad, Hercules, CA). Before immunoprecipitation bovine serum albumin was added to the precleared lysates (final concentration, 0.5%). Equal amounts of protein from lysates were always used for immunoprecipitation and Western blotting. Incubation of tissue lysate with chicken anti-human VEGF (a gift from Dr. Don Senger, Beth Israel Deaconess Medical Center, Boston, MA) followed by protein-G Sepharose beads was performed for 2 hours at 4°C. Immunoprecipitates were washed twice with Nonidet P-40 buffer (50 mmol/L Tris-HCl, pH 7.5, 10% glycerol, 150 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid, 1% Nonidet P-40 and sodium vanadate). Immunoprecipitation of KDR was performed using a monoclonal antibody (Chemicon, Temecula, CA). For these experiments, endometrial samples were solubilized in a similar buffer to the one previously described for VEGF extraction, but containing 1% Triton X-114 for isolation of membranes. Further purification of membrane fractions was subsequently done by ultracentrifugation on sucrose gradients. Anti-phosphotyrosine PY-20 antibody (BD Transduction Labs., San Jose, CA) was used on Western blots and an antibody to the N-terminal sequence of sFLT-1 was used on immunoprecipitation and Western analysis. Immunodetection in all cases was performed by incubation with specific peroxidase-conjugated secondary antibodies followed by enhanced chemiluminescence (Pierce, Rockford, IL).
Immunocytochemistry
Tissue samples were fixed in 4% paraformaldehyde in PBS for 1 to 2 hours, embedded in paraffin, and sectioned at 5 µm. Sections were cleared, rehydrated, washed, and blocked with 1% goat serum. Tissue sections were subsequently incubated with a monoclonal antibody against Ki-67 (Immunotech, Westbrook, ME) that detects nuclear-associated antigen of proliferating cells and is absent in resting cells (G0) but is present through the rest of the cell cycle.24 After several washes in PBS, sections were incubated with anti-Von Willebrand factor (DAKO, Glostrup, Denmark). The sections were washed and incubated simultaneously with two secondary antibodies: anti-rabbit alkaline phospatase-conjugated and anti-mouse peroxidase-conjugated. Development was performed according to the manufacturer’s protocol (Vectors Laboratories, Burlingame, CA). Quantification of endothelial proliferating cells was accomplished using ImagePro 3.1 software, scoring of positive nuclei in the stained (red) vessels was assessed within 1,000 endothelial nuclei per section.
Statistics
All categorical data are presented as a mean ± SD when repeated measures were done. Assuming normal distributions, data were analyzed by one-way analysis of variance, followed by either t-test with Dunnett test for comparisons between specific groups, or the Student-Newman-Keuls test for multiple comparisons between groups.25 Statistical analysis was performed by In-Stat software (Graph Pad Software) for Macintosh.
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Results |
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. These included early menstrual, late
menstrual, early proliferative, late proliferative, early secretory,
and late secretory endometrium. As per study design, the late
proliferative phase group had significantly higher estradiol
(P < 0.001) and luteinizing hormone
(P < 0.01) levels than menstrual or early
proliferative phases. Additionally, the early secretory and late
secretory phases had significantly higher progesterone levels
(P < 0.001 and P < 0.05,
respectively) than other phases of the cycle, indicative of ovulation.
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.
Although VEGF mRNA is constitutively expressed at low levels throughout
the menstrual cycle, a significant 9.3-fold induction was detected in
the menstrual phase (P < 0.001) (Figure 1B)
.
Because of the sharp induction of VEGF mRNA, the menstrual phase was
dropped out of the analysis of variance to analyze steady-state levels
in the proliferative and secretory phases. After excluding
the menstrual phase, a significant but small 1.6-fold induction was
found in the late secretory phase (P < 0.05) as
compared to all other phases in the cycle in agreement with previous
studies.13
To evaluate the potential role of hypoxia in
the menstrual phase endometrium, we hybridized the same Northern blot
with a cDNA probe for the glucose transporter GLUT-1, a marker of
anaerobic metabolism and an ischemia-induced gene.26
GLUT-1 is similarly co-expressed with a significant 9.6-fold induction
of steady-state mRNA levels in the menstrual phase
(P < 0.01) (Figure 1, A and B)
. The induction
of GLUT-1 signifies the hypoxic state of the endometrium and the
potential contribution of hypoxia to the regulation of VEGF during the
menstrual phase. We next evaluated the steady-state mRNA expression
pattern of the VEGF-related proteins, VEGF-B, VEGF-C, and PlGF (Figure 1C)
. Because of the low-level mRNA expression, we performed poly(A+)
enrichment of 50 µg of total RNA in a subset of samples
(n = 4/group). In Figure 1
(C and D), VEGF-B,
VEGF-C, and PlGF were shown to be constitutively expressed at low
levels throughout the endometrial cycle and did not seem to be
regulated by either hypoxia or sex steroids, as per their lack of
association with any particular phase. PlGF was highly expressed in the
placental specimen used as control. There was no significant difference
in steady-state RNA levels of any of these factors among endometrial
groups.
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Figure 2A
shows the induction of VEGF
mRNA in human endometrial stromal cells after exposure to hypoxic
conditions (3% O2). An increase in VEGF mRNA was
detected as early as 6 hours (1.5-fold, P < 0.05).
Maximal induction (2.4-fold more than control) was seen by 48 hours
(P < 0.001). GLUT-1 mRNA was equally induced by
hypoxia in vitro, demonstrating a 3.3-fold increase more
than control at 48 hours (data not shown). An increase of VEGF mRNA
levels on hypoxia has been shown to be variable among tumor
cells.27
While performing these studies, we evaluated the
relative ability of endometrial fibroblasts to respond to hypoxia by
their ability to induce VEGF compared to fibroblasts isolated from
lung, skin, breast, bone marrow, and heart. Interestingly, endometrial
fibroblasts were the most responsive followed by lung, heart, skin,
bone marrow, and breast (data not shown).
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, our
in vitro conditions either did not reproduce the
degree of hypoxia/ischemia typical of the menstrual phase in
vivo or additional confounding stimulators were missing in these
experimental conditions. Hypoxic experiments were repeated under a
variety of steroid treatments, including after steroid withdrawal
in vitro, to best reproduce the physiological sex steroid
withdrawal typical of the menstrual phase. Northern analysis was
conducted for VEGF expression after exposure to 3 days of estradiol (10
nmol/L) and progesterone (10 µmol/L), followed by a steroid-free
withdrawal period. No relative change was seen under normoxic
conditions throughout a 48-hour period, revealing no direct effect of
steroid withdrawal on VEGF mRNA levels (data not shown). Similarly,
steroid withdrawal under hypoxic conditions did not seem to potentiate
steady-state VEGF mRNA induction over the effect of hypoxia alone.
Incubation with estradiol under normoxic conditions resulted in a
modest, but significant 1.6-fold induction of VEGF mRNA
(P < 0.05) at 6 hours, although mRNA levels
quickly returned to baseline after estradiol withdrawal. Incubation
with progesterone resulted in a 1.3-fold induction of VEGF mRNA at 6
hours, but this was not significant (data not shown). These data
support previous reports that indicate a consistent but low
contribution of steroids in VEGF regulation in the
endometrium28,29
We next hypothesized that additional cytokines could contribute to the
enhancement in VEGF expression seen during the menstrual phase. Two
cytokines in particular (TGF- and IL-1) have been implicated in the
regulation of VEGF mRNA in several cell types.30,31
Interestingly, both cytokines were increased in menstrual endometrium
and down-regulated during the remaining phases of the endometrial cycle
(Figure 2C)
following a profile similar to that of VEGF mRNA. These
data temporally placed these cytokines as potential regulators of VEGF
mRNA in this tissue. Neither TGF- nor IL-1ß was increased on
hypoxic treatment of stromal cells (data not shown). It is likely that
the major source of TGF- and IL-1ß in the endometrium is
inflammatory cells, abundant in the menstrual endometrium, as compared
to other phases of the cycle.32
Induction of VEGF mRNA by IL-1ß in vitro was dose- and
time-dependent, with a maximal 4.2-fold induction greater than control
after a 4-hour incubation of human endometrial stromal cells with
IL-1ß (10 ng/ml) (Figure 3, A and C)
.
Incubation of endometrial stromal cells with TGF- (50 ng/ml)
throughout a 48-hour time course resulted in a maximal 2.1-fold
induction of VEGF mRNA by 3 hours. Transcript levels returned to
baseline by 48 hours (Figure 3, B and D)
. Induction of VEGF mRNA by
TGF- was dose-dependent with a maximal 4.7-fold induction at 200
ng/ml (data not shown). TGF- induction was inhibited by
pre-incubation with anti-TGF- antibodies (Figure 3E)
. Similarly the
effect mediated by IL-1ß was reversed if the cytokine was
pre-incubated with neutralizing antibodies (Figure 3E)
. As an important
control, we found that fibroblast growth factor-2, a cytokine also
present in the human endometrium was not able to enhance VEGF
transcript levels (Figure 3F)
.
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, IL-1ß, and hypoxia increased VEGF mRNA
levels 15-fold (Figure 4A)
. The effect
mediated by these three factors together was clearly greater than the
sum of each alone. Hypoxia also potentiated the effects mediated by
TGF- (Figure 4B)
and IL-1ß independently (data not shown). To best
understand the relationship between hypoxia and growth factor
regulation, we performed experiments in which stromal cultures were
first subjected to hypoxia for 24 hours, followed by normoxia for 16
hours, and then treated with cytokines for an 16 additional hours.
Levels of VEGF were compared to similar cultures that were first
maintained in normoxia for 24 hours, followed by hypoxia for 16 hours,
and then treated with cytokines in hypoxia (Figure 4C)
. The increase of
VEGF mRNA by hypoxia was shown to be transient and returned to baseline
on normalization of O2 levels (Figure 4C
, lane
C). Previous exposure to hypoxia did not alter the response of VEGF
transcript levels to TGF-. Treatment with this cytokine alone
induced VEGF levels by 2.5- to 3.5-fold, as shown in Figure 3
. Again, a
small increase was seen when stromal cells were treated simultaneously
with TGF- and IL-1ß even with previous exposure to hypoxia (Figure 4C, H
+ N). In contrast, constant hypoxic conditions showed an additive
effect on treatment with TGF- or IL-1ß and clear potentiation when
both of these cytokines were used in combination (Figure 4C, N
+ H).
These results indicate that hypoxia can prolong the transient
stimulation mediated by cytokines and provides further evidence for an
additive effect of up to 15-fold when both cytokines are used in
combination. Together, the temporal pattern of expression of TGF-
and IL-1ß suggests that these cytokines in combination with hypoxia
are likely responsible for the peak and sustained levels of VEGF
transcripts in the menstrual phase of the endometrial cycle.
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. VEGF detected by ELISA was
normalized to total protein present in media. Hypoxia was associated
with a 2.9-fold increase in VEGF protein more than normoxic controls at
48 hours. The addition of sex steroids did not significantly increase
VEGF protein levels more than normoxic conditions, or augment the
hypoxic induction in VEGF protein. Nonetheless, an impressive increase
in protein was seen with the combination hypoxia, TGF-, and IL-1ß,
confirming the data obtained by Northern analysis.
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. FLT-1 was increased by 2.8-fold
(P < 0.01) and KDR by 3.2-fold
(P < 0.01) in the menstrual as compared to
other phases of the cycle. This pattern follows the one described
earlier for VEGF (Figure 1)
, yet these data did not provide further
information as to the signaling status of these receptors.
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. KDR was increased in the
menstrual phase and early in the proliferative phase (Figure 7A)
. The
peak during the menstrual phase was parallel to the high levels of
VEGF. Consequently, we had anticipated a high phosphorylation state for
this receptor during the menstrual phase. Interestingly,
phosphorylation only peaked late in the menstrual phase with lower
sustained phosphorylation levels during the proliferative phase (Figure 7A
, PY antibody). The pattern was reproduced in all three independent
samples evaluated. The reason for this lack of correlation between the
peaks of KDR protein expression and phosphorylation levels implies an
additional tier of regulation in receptor signaling. Because sFLT-1 has
been implicated in the negative regulation of VEGF receptor pathway by
binding to the growth factor and preventing interaction with its
receptors,12
we asked whether this mechanism of regulation
might also be present in the human endometrium. Indeed, sFLT-1 was
immunoprecipitated from endometrial samples (Figure 7B)
. Major
expression of sFLT-1 was detected in early menstrual specimens, but
protein was also seen in the late secretory phase. The pattern of
sFLT-1 expression provided a temporal correlation with the decrease of
KDR phosphorylation in the early menstrual phase. Additional supporting
data were obtained when anti-VEGF antibodies were used in early
menstrual samples to immunoprecipitate the growth factor.
Immunoprecipitated VEGF was bound to sFLT-1 (Figure 7C)
. These results
provided evidence that indeed during the early menstrual phase, at
least some VEGF is bound to sFLT-1. This effect was transient, because
sFLT-1 was not detected in late menstrual phase (Figure 7C
, lane 2) and
consistent with the low/undetectable levels of sFLT-1 in the late
menstrual phase (Figure 7B)
.
|
. The menstrual (day 3) biopsy seen in
Figure 8, A and B
, reveals a significant portion of viable,
proliferating endothelial cells in the basal layer. Proliferation was
also detected in the proliferative (Figure 8, C and D)
and secretory
phases (Figure 8E)
. Quantification of proliferating endothelial cells
revealed that these cells proliferate throughout the cycle, as
previously shown,9
nonetheless, detailed evaluation of the
menstrual phase showed a statistically significant increase late in the
menstrual phase and early in the proliferative phase (Figure 8E)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß are most
likely responsible for the induction and sustained levels of VEGF
during menstruation. Finally we demonstrate that the KDR
phosphorylation pattern does not follow the pattern of total receptor
levels. Interestingly, phosphorylation of KDR is lower when sFLT-1 is
present. We showed that this molecule can be found in association with
VEGF in endometrial specimens providing a mechanism by which VEGF
receptor phosphorylation might be regulated during the endometrial
cycle.
Given the importance of oxygen tension as a regulator of blood vessel
growth and development,23,35
the contribution of
physiological hypoxia associated with menstruation and its role in
regulating endometrial angiogenesis has been poorly explored. Ischemic
cellular stress leads to induction of hypoxia-inducible factor
(HIF-1) a transcription factor that regulates several genes,
including VEGF.23,36
Aside from an increase in
transcription rates through HIF-1, hypoxia also promotes increases
in VEGF mRNA stability.37
The overall effect is a
sustained elevation in secreted VEGF protein with increased endothelial
cell survival and proliferation, granted the appropriate receptors on
the endothelial cell surface are expressed. Taking this background into
consideration, it is not at all surprising that VEGF levels were raised
in menstrual tissue. Our study shows that under hypoxic conditions
endometrial stromal cells increase VEGF transcript levels. Our findings
are in agreement with two recent studies that have examined the
contribution of hypoxia to VEGF mRNA increase by endometrial
cells.38,39
However, alone, hypoxia could not account for
the marked expression of VEGF seen in the menstrual phase. In fact,
both our studies and the one reported previously39
found
no more than a 3.4-fold increase in VEGF levels in culture. Therefore
contributing cytokines and/or hormones are likely to aid in the
significant increase seen in the menstrual basal endometrium.
Two major cytokines have been implicated as potential regulators of
VEGF expression in inflammatory states: TGF- and IL-1ß. In
psoriasis, an inflammatory condition associated with overexpression of
VEGF, the increased VEGF levels are most likely mediated by TGF-,
which has been shown to increase VEGF expression in epidermal
keratinocytes in vitro.40,41
IL-1ß has been
also shown to up-regulate VEGF mRNA in Kaposi’s sarcoma spindle
cells31
and to increase VEGF mRNA levels in aortic smooth
muscle cells.34
We sought out TGF- and IL-ß as
candidate cytokines for VEGF regulation in human endometrium. TGF-
has been described in endometrium, with intense immunostaining noted
around the spiral arterioles.42
IL-1ß protein and
transcript have also been localized to the endothelium of the spiral
arterioles.43
Our studies indicated that both TGF- and
IL-1ß regulate VEGF mRNA in human endometrial stromal cells. The
combined effects of hypoxia, TGF-, and IL-1ß on VEGF mRNA in
cultures of stromal cells were comparable with the increased levels of
VEGF mRNA seen in the menstrual endometrium. Additional
pro-inflammatory cytokines, in particular TNF- and IFN-
have also
been examined for their ability to affect VEGF transcripts in
endometrial stromal cells. These cytokines do not increase VEGF
expression in stromal cultures (M. L. Iruela-Arispe, unpublished
observations),17
in fact, one study reported a decrease in
VEGF transcripts after 6 hours of stromal cell exposure to
IFN-.44
In our endometrial stromal cultures, the positive contributions of estradiol and progesterone to VEGF up-regulation were modest, with maximal 1.6- to 1.8-fold increases, as has been reported by others.13,28,29,45 Although VEGF up-regulation in the secretory phase may be explained by progesterone effects, it is unlikely sex steroids play a direct role on VEGF regulation during postmenstrual repair as circulating estrogen and progesterone levels are physiologically low at this point in the cycle.
Additional members of the VEGF cytokine family were constitutively expressed at low levels during the menstrual cycle and do not seem to be either hypoxia- or steroid-regulated. These data concur with previous findings that VEGF-B and VEGF-C were not increased by hypoxia in vitro in cell types other than fibroblasts.46 Constitutive expression of additional VEGF family members may provide a positive angiogenic stimulus for the endometrium during all phases of the cycle. Hence, the potential for endometrial blood vessel growth is always present.9,47 Regulation of endometrial angiogenesis may then occur through a rise in angiogenic inhibitors. We have previously shown that thrombospondin-1, an inhibitor of angiogenesis, is increased during the early secretory phase of the human endometrial cycle, and is regulated by progesterone in vitro.21 It is possible that steroid contribution to the vascular repair in endometrium occurs predominantly through the regulation of inhibitory pathways, eg, via thrombospondin-1.
Relevant to our understanding of VEGF function during the endometrial cycle are also the patterns displayed by its receptors: FLT-1 and KDR. Our findings indicate that both receptors are present mostly during the menstrual and proliferative phases, decreasing thereafter. Others have reported presence of full length FLT-1 and KDR at "almost constant levels" throughout the endometrial cycle by RT-PCR,48 however the menstrual phase was not part of these analyses. As for the distribution of VEGF receptors by immunocytochemistry, expression of FLT-1, in contrast to KDR, was observed primarily in dilated capillaries during the premenstrual period.49 Interestingly, recent studies have co-localized KDR in stromal cells of the superficial endometrial zones during the premenstrual phase50 and in epithelial cells.49 The functional significance of this expression remains unclear. Considering that our Northern analysis included the entire endometrium, it is likely that we were also detecting receptor-expressing nonendothelial cells.
In addition, and perhaps more pertinent, to VEGF regulation are the levels of sFLT-1. The soluble form of FLT-1 was identified initially in the conditioned media of human umbilical vein endothelial cells.51,52 This protein was shown to act as a dominant-negative effector by forming inactive heterodimers with transmembrane receptors and by sequestering VEGF.53 sFLT-1 has been found in the conditioned media of tumor cells, endothelial cells, and in amniotic fluid.54 More directly relevant to our findings, sFLT-1 has been found in placenta and in the serum of pregnant women indicating that regulation of VEGF by sFLT-1 is an important event in implantation and the maintenance of pregnancy.55 A recent study has described the presence of sFLT-1 by RT-PCR in endometrium mostly during the secretory phase.48 Our data agrees with these findings, at the protein level, but in addition we showed a significant increase during the early menstrual phase. More interestingly, we found a correlation between decreased KDR phosphorylation and presence of sFlt-1. Given the strong body of literature12,53,55 implicating sFLT-1 in the sequestration of VEGF from its transmembrane receptors, it is tempting to speculate that expression of sFLT-1 during the menstrual phase might modulate KDR receptor signaling by retarding the wave of vascular repair. In addition, we found that VEGF is bound to sFLT-1 early in the menstrual phase. Consistent with these correlations, we found a small, yet statistically significant, peak of endothelial cell proliferation that coincides with time of KDR phosphorylation. Clearly further experimentation and additional mechanistic studies will be required to support this model.
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Supported by the National Institutes of Health (grant R29C65624 and RO3CA70559-02) and by the Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Beth Israel Deaconess Medical Center, Boston.
In memory of Joseph F. Mortola.
This work was presented in part at the 44th Annual Meeting of the Society for Gynecologic Investigation, San Diego, California, 1997.
Current address of M. D. Graubert: Palmetto Fertility Center of South Florida, Miami, FL 33016.
Accepted for publication January 12, 2001.
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, M versus all other groups
(P < 0.01). The 1.6-fold
induction of VEGF mRNA in LS phase is significantly greater than EP,
LP, ES, after removing M phase from the analysis of variance
(P < 0.05).
C: Northern blot of
poly(A+)-enriched RNA
(isolated from 50 µg of total
RNA) extracted from the same stages as in
A and hybridized to VEGF-B, VEGF-C, and PlGF cDNA probes.
D: Northern blots from independent biopsies
(n = 4) were scanned by
densitometry and normalized to signal from 36B4 mRNA
(ribosomal-associated
protein). Fold induction values between groups
expressed as mean ± SD. Because of large variability in
individual samples, no significant difference was detected between
groups.
, hypoxia
(3%) in the presence of
IL-1ß (10 ng/ml) and
TGF-
