Human Reproduction, Vol.27, No.2 pp. 523–530, 2012
Advanced Access publication on November 28, 2011 doi:10.1093/humrep/der405
ORIGINAL ARTICLE Reproductive biology
Hypoxic stress simultaneously
stimulates vascular endothelial growth
factor via hypoxia-inducible factor-1a
and inhibits stromal cell-derived factor-1
in human endometrial stromal cells
Tomoko Tsuzuki, Hidetaka Okada*, Hisayuu Cho, Shoko Tsuji,
Akemi Nishigaki, Katsuhiko Yasuda, and Hideharu Kanzaki
Department of Obstetrics and Gynecology, Kansai Medical University, 2-3-1 Shinmachi-cho, Hirakata, Osaka 573-1191, Japan
*Correspondence address. Tel: +81-72-804-010, ext: 3660; Fax: +81-72-804-2865; E-mail: [email protected]
Submitted on August 23, 2011; resubmitted on October 19, 2011; accepted on October 27, 2011
background: Hypoxia of the human endometrium is a physiologic event occurring during the perimenstrual period and the local stimulus for angiogenesis. The aim of this study was to investigate the effects of hypoxic stress on the regulation of vascular endothelial growth
factor (VEGF) and stromal cell-derived factor-1 (SDF-1/CXCL12), and the potential role of hypoxia-inducible factor-1a (HIF-1a) in the
endometrium.
methods: Human endometrial stromal cells (ESCs, n ¼ 22 samples) were studied in vitro. ESCs were cultured under hypoxic and normoxic conditions and treated with cobalt chloride (CoCl2; a hypoxia-mimicking agent) and/or echinomycin, a small-molecule inhibitor of
HIF-1a activity. The mRNA levels and production of VEGF and SDF-1 were assessed by real-time PCR and ELISA, respectively. The
HIF-1a protein levels were measured using western blot analysis.
results: Hypoxia simultaneously induced the expression of mRNA and production of VEGF and attenuated the expression and production of SDF-1 from ESCs in a time-dependent manner. Similar changes were observed in the ESCs after stimulation with CoCl2 in a dosedependent manner. CoCl2 significantly induced the expression of HIF-1a protein, and its highest expression was observed at 6 h. Echinomycin inhibited hypoxia-induced VEGF production without affecting the HIF-1a protein level and cell toxicity and had no effect on SDF-1
secretion (P , 0.01).
conclusions: Hypoxia simultaneously acts to increase VEGF via HIF-1a and to decrease SDF-1 in a HIF-1a-independent manner in
ESCs. These results indicate a potential mechanism for the action of hypoxic conditions that could influence angiogenesis in the human
endometrium.
Key words: endometrial stromal cells / hypoxia / hypoxia-inducible factor-1a / vascular endothelial growth factor / stromal cell-derived
factor-1
Introduction
The human endometrium is a dynamic tissue that undergoes regular
cycles of menstruation, menstrual repair, proliferation and secretory
differentiation. Angiogenesis is important in the cyclical repair and
regeneration of the endometrium during the menstrual cycle (Aberdeen et al., 2008). Hypoxia in the endometrial tissues is a physiologic
event that occurs during the premenstrual period. Local hypoxia is a
major regulator of endometrial angiogenesis and remodeling during
menstruation (Salamonsen, 2003). The complex processes of angiogenesis are presumably tightly regulated by multiple system controls
that can be switched on and off within a short period.
Vascular endothelial growth factor (VEGF) is a key regulator of
angiogenesis and vascular function. Recent studies have reported the
role of VEGF in the early angiogenic processes associated with postmenstrual regeneration of endometrium (Fan et al., 2008). VEGF is
essential for the rapid burst of angiogenesis that occurs during postmenstrual repair and early proliferative phases in the primate
& The Author 2011. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
For Permissions, please email: [email protected]
524
endometrium and further plays a role in re-epithelialization of the
endometrium (Fan et al., 2008). Stromal cell-derived factor-1
(SDF-1, also known as CXCL12) and VEGF play important roles in
angiogenesis. In the vasculature, SDF-1 functions as a potent inducer
of angiogenesis, stimulating endothelial cell proliferation and cell
survival (Salcedo et al., 1999). VEGF and SDF-1 interact synergistically
to promote the functions of vascular endothelial cells, such as cell
migration, cell survival and changes in gene expression (Salcedo
et al., 1999; Bachelder et al., 2002; Nishigaki et al., 2010). VEGF and
SDF-1 are present in the human endometrium (Sugino et al., 2002;
Ruiz et al., 2010). SDF-1 has been shown to be up-regulated in
many damaged tissues as part of the injury response and is thought
to channel stem and progenitor cells to promote repair (Burger and
Kipps, 2006). As shown in several recent studies on SDF-1 for
human endometrial function, SDF-1 may play crucial roles in endometrial proliferation, embryo implantation and leukocyte recruitment to
the endometrium (Dominguez et al., 2003; Wu et al., 2005; Tsutsumi
et al., 2011). In view of the pleiotropic functions, SDF-1 and VEGF are
thought to significantly influence angiogenesis and endometrial vascularization. Therefore, in this study, we investigated VEGF and SDF-1
and their potential functions as the angiogenic factors that contribute
to the regulation of angiogenesis and proliferation of the human
endometrium.
Previous studies suggested that the expression of VEGF and SDF-1
in the endometrium is regulated by female sex steroids (Okada et al.,
2009; Tsutsumi et al., 2011). Estradiol (E2) induced mRNA and protein
production of VEGF and SDF-1 in primary cultures of human endometrial stromal cells (ESCs), whereas progesterone had inhibitory
effects on E2-induced VEGF and SDF-1 production (Okada et al.,
2011). On the other hand, hypoxia is believed to be an important
regulator of angiogenesis and stimulates mRNA expression and
production of VEGF and SDF-1 in other tissues (Martin et al., 2010;
Dong et al., 2011).
Hypoxia-inducible factor-1a (HIF-1a) is a transcription factor
known to play a major role in the cellular response to hypoxia.
HIF-1a protein is suppressed in cells under normoxic conditions
(20 –22% O2) and its expression is rapidly induced by hypoxic conditions (1 –2% O2). In normoxia, HIF-1a is post-translationally hydroxylated by prolyl hydroxylases in the oxygen-dependent degradation
domain. This interaction results in ubiquitination and subsequent
proteasome degradation of HIF-1a. Hypoxia inhibits the hydroxylation
steps, which prevents the interaction of HIF-1a with the von Hippel –
Lindau tumor suppressor protein, which recruits a ubiquitin –ligase
complex, and the subsequent ubiquitination and degradation of
HIF-1a (Ivan et al., 2001; Jaakkola et al., 2001). VEGF and a number
of other angiogenic genes have been shown to be transcriptionally
regulated by HIF-1a (Semenza, 2011). In the human endometrium,
HIF-1a protein is abundant in glandular and stromal cells in the functional layer during the late secretory and menstrual phases (Critchley
et al., 2006). However, the regulation of VEGF and SDF-1 via HIF-1a
activation under hypoxic condition in the human endometrium
remains poorly understood.
We hypothesized that hypoxia is involved in the regulation of VEGF
or SDF-1 in the endometrium. In the present study, we evaluated the
effects of hypoxia on the expression of VEGF and SDF-1 by using ESCs
in culture and investigated the potential role of HIF-1a in the regulation of these angiogenic factors under hypoxic conditions.
Tsuzuki et al.
Materials and Methods
Tissue collection and culture of ESCs
All human tissues were obtained using a protocol for the protection of
human subjects approved by the ethical committee of Kansai Medical
University, Japan, together with informed consent from all patients and
in accordance with the Declaration of Helsinki. Human endometrial
tissues were obtained from 22 patients, aged 34 – 47 years, in the proliferative phase, with regular menstrual cycles, who underwent hysterectomies
for the treatment of myoma uteri without hormonal therapy. ESCs were
purified by the standard enzyme digestion method as described previously
(Okada et al., 2005). ESCs were cultured in Dulbecco’s modified Eagle’s
medium (DMEM)/F-12 medium supplemented with 10% fetal calf serum
(FCS) (HyClone, Logan, UT, USA), 100 IU/ml penicillin and 100 mg/ml
streptomycin (Invitrogen, Carlsbad, CA, USA) at 378C under a humidified
atmosphere of 5% CO2 in air. The culture medium was replaced 30 min
after plating to reduce epithelial cell contamination. The percentage of
vimentin-positive cells in confluent ESCs was .99% by immunohistochemical staining as described previously (Okada et al., 2001).
Hypoxic stimulus and reagents for ESCs
After passage 0 – 1 when ESCs were nearly confluent, cells were trypsinized and re-plated in 6-well plates (1 × 106 cells/well) for real-time
PCR analyses, 24-well plates (2 × 105 cells/well) for ELISA. ESCs were
cultured in 10% FCS-supplemented DMEM/F-12 medium under hypoxic
conditions (2% O2, 5% CO2 and 93% N2) or normoxic conditions (5%
CO2 and 95% air), resulting in an oxygen concentration of 20%. In
drug-induced hypoxic experiments, ESCs were cultured in 10%
FCS-supplemented medium containing various amounts of cobalt chloride
(CoCl2, a hypoxia-mimicking agent; 0.1 – 100 mmol/l; Wako, Osaka,
Japan), echinomycin (a HIF-1a inhibitor; 0.1 – 100 nmol/l; ENZO Life
Sciences, NY, USA) and/or dimethylsulfoxide as vehicle control under
5% CO2 in air. The supernatant was collected after stimulation and
stored at 2808C until assayed. Each experiment was repeated at least
three times with different cell preparations.
RNA extraction and real-time PCR analysis
Total RNA was isolated from cultured ESCs using an RNeasy Minikit
(Qiagen GmbH, Hilden, Germany) according to the manufacturer’s
instructions. Quantitative real-time PCR was performed using the SYBR
green I nucleic acid Gel Stain (Roche, Diagnostics GmbH, Mannheim,
Germany) as described previously (Nakamoto et al., 2005). Elongation
factor-1a (EF-1a) was used as an internal control, as it is a valid reference
‘housekeeping’ gene for transcription profiling, which is also used for realtime PCR experiments. Forward (F) and reverse (R) primers used in this
study were as follows: VEGF, 5′ -CAGGACCATGAACTTTCTGC-3′ (F)
and 5′ -CCTCAGTGGGCACACACTCC-3′ (R); SDF-1, 5′ -TCGTGCT
GACCGCGCTCTGCCTCA-3′ (F) and 5′ -TCTGAAGGGCACAGTTT
GGAGTGT-3′ (R); EF-1a, 5′ -TCTGGTTGGAATGGTGACAACATGC-3′
(F) and 5′ -AGAGCTTCACTCAAAGCTTCATGG-3′ (R). PCR of all standards and samples was performed using duplicate reactions, after which a
melting curve analysis was performed to monitor PCR product purity. In
order to eliminate the possibility of contamination with genomic DNA
during extraction of total RNA, a control reaction with each primer pair
was performed at the same time under identical conditions without
reverse transcription, and no amplification was detected.
VEGF and SDF-1 assay by ELISA
The release of VEGF and SDF-1 into cell culture supernatants was
assessed using commercially available sandwich ELISA kits (Quantikine
525
Hypoxia affects the angiogenic factors
ELISA kit; R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Intra-assay and inter-assay coefficients of variation
in cell culture supernatants were 2.2 and 8.9% for VEGF and 3.3 and
7.2% for SDF-1, respectively.
Lactate dehydrogenase release analysis
Measurement of lactate dehydrogenase (LDH) release was conducted to
examine the effect of drug treatment on cell viability. After exposure to
various treatments for 48 h, conditioned media were collected. The
release of LDH into culture media was measured using a specific Cytotoxicity Detection kit (Roche) according to the manufacturer’s instructions.
Absorbance data were obtained with a 490 nm filter and a 620 nm filter
as reference wavelength. LDH release as high (positive) control was determined by cell culture media containing 5% Tween 20.
Western blot analysis
For analysis of HIF-1a protein levels, cultured cells treated with CoCl2 and
echinomycin were homogenized in lysis buffer containing Mammalian
Protein Extraction Reagent (Thermo Fisher Scientific Inc., Rockford, IL,
USA) and protease inhibitor cocktail. Samples were subjected to 7.5%
sodium dodecyl sulfate– polyacrylamide gel electrophoresis and transferred to Immun-Blot polyvinylidene diflouride Membrane (Bio-Rad,
Laboratories, Inc., Hercules, CA, USA). Non-specific-binding sites were
blocked with 10% skim milk powder in Tris-buffered saline for 30 min.
Blots were then incubated for 1 h at room temperature with rabbit monoclonal HIF-1a antibody (1:1000; Epitomics, Inc., Burlingame, CA, USA) or
mouse monoclonal b-actin antibody (1:5000; Sigma-Aldrich Corp.,
St. Louis, MO, USA) as a primary antibody, and anti-rabbit immunoglobulin (Ig)G peroxidase-labeled secondary antibody (1:5000; GE Healthcare
Life Science, St. Giles, UK) or anti-mouse IgG peroxidase-labeled secondary antibody (1:10000; GE Healthcare Life Science) as a secondary
antibody. Immune complexes were visualized by enhanced chemiluminescence plus Western Blotting Detection Reagents (GE Healthcare Life
Science). We analyzed the protein levels using ImageJ software and visualized quantitatively. ImageJ is a public domain image-processing program
developed by the National Institutes of Health and is freely available at
http://rsbweb.nih.gov/ij. Fold increase was calculated by dividing the relative expression of HIF-1a by the relative expression of b-actin.
Statistical analysis
Data are expressed as mean + SEM. Results were analyzed with a statistical software package, StatView II version 4.0 (Abacus Concepts, Berkeley, CA, USA). Differences in the measured parameters across the
different groups were statistically assessed using analysis of variance with
repeated measurements, followed by Fisher’s protected least significant
difference, multiple range test. A level of P , 0.05 was considered statistically significant.
Results
Effects of hypoxia on mRNA expression and
protein secretion of VEGF and SDF-1 in ESCs
To characterize the effects of hypoxia on VEGF and SDF-1 mRNA
expression, we analyzed cultured ESCs under hypoxic (2% O2) or normoxic (20% O2) conditions for 48 h. Real-time PCR analysis demonstrated that hypoxia caused a significant increase in the levels of VEGF
mRNA expression (Fig. 1A). In contrast, SDF-1 mRNA expression was
significantly inhibited in the hypoxic condition (Fig. 1B). As shown in
Fig. 1C and D, the levels of VEGF protein significantly increased in
response to hypoxia, whereas those of SDF-1 were significantly attenuated. The temporal release of VEGF and SDF-1 from ESCs in
response to hypoxia is shown in Fig. 2. Hypoxia caused a significant
increase of VEGF production after 6 h of culture compared with normoxia, and this effect continued to increase until the end of the study
at 48 h. In contrast, SDF-1 production was significantly suppressed
after 24 h in response to hypoxia, and this effect continued until the
end of the study at 48 h. These findings suggest that hypoxia simultaneously stimulates VEGF production and inhibits SDF-1 production in
cultured ESCs.
Effects of CoCl2 on VEGF and SDF-1
secretion from ESCs
The effects of hypoxia on secretion of VEGF and SDF-1 were further
confirmed by culturing ESCs in the presence of CoCl2, which induced
environment-mimicking hypoxic conditions. ESCs were incubated with
various doses of CoCl2 for 48 h. As shown in Fig. 3A, 1 mM CoCl2 had
no effect, but VEGF production was induced by 10 mM CoCl2, and
100 mM CoCl2 strongly induced VEGF production. On the other
hand, CoCl2 significantly attenuated SDF-1 production from ESCs in
a dose-dependent manner, when compared with controls (Fig. 3B).
The maximal effect was observed at 100 mM, but a significant
decrease was observed at 0.1 mM. In order to exclude the potential
toxicity of CoCl2 on ESCs, we assessed cell viability using LDH
assay. CoCl2 at a concentration of 100 mM did not increase LDH
release from ESCs (data not shown). Therefore, in all subsequent
experiments, we chose the 100 mM concentration of CoCl2 to
mimic hypoxic conditions.
CoCl2 treatment increases HIF-1a protein
levels in ESCs
We then evaluated potential mechanisms that might explain the effect
of CoCl2 on VEGF and SDF-1 production in ESCs. It is well known that
CoCl2 can mimic HIF-1 activation through inhibition of HIF-1a degradation. By using western blot analysis, we determined whether the
addition of CoCl2 led to an increase in the levels of HIF-1a proteins
in the ESCs. As shown in Fig. 4A, CoCl2 significantly induces the
expression of HIF-1a protein levels at 2 and 6 h in ESCs, whereas
the addition of a vehicle to the culture medium had no effect. The
highest expression of HIF-1a protein was observed at 6 h, and the
levels had declined by 24 h. This experiment was repeated three
times with different batches of cells, and the results were similar
(Fig. 4B).
Effects of echinomycin on CoCl2-mediated
VEGF and SDF-1 secretion from ESCs
To demonstrate the role of HIF-1a in CoCl2-mediated VEGF and
SDF-1 protein production, we used echinomycin, a small-molecule
inhibitor of HIF-1a activity. It has been reported that echinomycin
specifically inhibits DNA binding of HIF-1a by directly interacting
with the core sequence within the hypoxia-response element (HRE)
of the target gene. We monitored the secretion of VEGF and SDF-1
under hypoxic conditions induced by CoCl2 with or without the
addition of echinomycin in ESCs. The 100 nM concentration of echinomycin almost completely inhibited the induction of VEGF under the
hypoxic condition induced by CoCl2 (Fig. 5A). However, the
526
Tsuzuki et al.
Figure 1 The effect of hypoxia on mRNA expression of VEGF (A) and SDF-1 (B) in ESCs. ESCs were cultured in normoxia (20% O2) or hypoxia
(2% O2) for 48 h. VEGF and SDF-1 mRNA levels were analyzed by real-time PCR analysis and calculated after normalization to EF-1a mRNA expression. The effect of hypoxia on VEGF (C) and SDF-1 (D) production in ESCs. The ELISA measured VEGF and SDF-1 concentrations in the culture
medium. The effect of normoxia on VEGF and SDF-1 production is assigned a potency of 100%. Results are combined data of four experiments
with different cell preparations and each value represents mean + SEM. *P , 0.01 versus normoxia.
echinomycin treatment did not affect the production of SDF-1
(Fig. 5B). The viability of cells, as measured by LDH release assay,
remained unchanged in ESCs treated with CoCl2 and/or echinomycin
in comparison with the untreated cells (Fig. 5C). Furthermore, to rule
out the possibility that the decrease of VEGF by echinomycin depends
on the down-regulation of HIF-1a protein levels, we performed
western blotting analysis to examine the HIF-1a protein levels in
ESCs treated with CoCl2 and/or echinomycin for 6 h. As shown in
Fig. 6A, CoCl2 induced HIF-1a protein accumulation, which did not
change with the addition of echinomycin. This experiment was
repeated three times with different batches of cells, and the results
were similar (Fig. 6B). These results show that echinomycin did not
affect HIF-1a protein levels and inhibition of hypoxic induction of
VEGF production. In conclusion, our results reinforced the hypothesis
that HIF-1a plays a major role in regulating the expression of VEGF
under hypoxic conditions in ESCs.
Discussion
The present study shows that hypoxia simultaneously induces the
expression of mRNA and production of VEGF and attenuates the
expression and production of SDF-1 in human ESCs. In addition,
similar changes in VEGF and SDF-1 were observed in ESCs with
CoCl2 stimulation, a chemical hypoxia-mimicking agent, which prevents the proteasomal degradation of HIF-1a proteins to create a
hypoxia-like condition. CoCl2 simply reverses the high level of
degradation induced by 20% O2, restoring the presence of a pool of
endogenous HIF-1a within ESCs. Our study shows that 100 mM
CoCl2 is sufficient to increase the HIF-1a level and VEGF production
in ESCs but concentrations of CoCl2 up to even 500 mM have been
used in studies of HIF-1a (Cho et al., 2005; Ardyanto et al., 2006).
It has been demonstrated that hypoxia stimulated VEGF mRNA
expression in ESCs (Popovici et al., 1999; Sharkey et al., 2000),
which is consistent with our data. These studies suggest that the
effect of hypoxia on VEGF production from ESCs might have physiologic relevance in the process of menstruation and in postmenstrual
endometrial repair and angiogenesis. To the best of our knowledge,
our study is the first to demonstrate that hypoxic stress inhibits
SDF-1 in human ESCs. These findings are surprising because hypoxia
has been shown to induce the production of several angiogenic stimulators, such as VEGF and SDF-1, in endothelial cells or cancer cells
(Schioppa et al., 2003; Takenaga, 2011). Taking into account our
present findings, ESCs are suggested to be able to control physiologic
angiogenesis in the endometrium by regulating the production of
angiogenic factors.
HIF-1a plays a critical role in cellular responses to hypoxia. HIF-1a
activation by the hypoxic microenvironment contributes to the induction or repression of the activity of genes involved in different cellular
functions, such as cell survival, oxygen homeostasis, proliferation,
angiogenesis, glucose metabolism and apoptosis (SzablowskaGadomska et al., 2011). Both studies in cultured cells and animal
models have demonstrated that HIF-1a was involved in the increased
Hypoxia affects the angiogenic factors
Figure 2 Time course of VEGF (A) and SDF-1 (B) secretion from
ESCs under the hypoxic condition. ESCs were cultured in normoxia
(20% O2) or hypoxia (2% O2) for 2, 6, 24 and 48 h. Results are representative data of four experiments with different cell preparations
and each value represents mean + SEM. *P , 0.01 versus normoxia.
expression of VEGF and SDF-1 in response to hypoxia and ischemia
(Yamakawa et al., 2003; Ceradini and Gurtner, 2005). HIF-1a has
been shown to directly bind to HREs in the promoters of the genes
encoding VEGF. In the human endometrium, HIF-1a protein is
expressed with increasing intensity from the premenstrual to the
menstrual phase but no HIF-1a immunoreactivity was detected in
endometrial biopsies from the proliferative phase (Critchley et al.,
2006). The temporal and spatial distribution of HIF-1a protein
would thus seem to be related to the process of menstruation. In
fact, hypoxia has been shown to induce VEGF mRNA expression via
HIF-1a activation in the human endometrium (Yoshie et al., 2009).
The present study demonstrates that SDF-1 production is attenuated by hypoxia in human ESCs. Our previous studies showed that
SDF-1 and VEGF were up-regulated by E2 (Okada et al., 2009,
2011; Tsutsumi et al., 2011) and that progesterone inhibited
E2-induced SDF-1 and VEGF expression in ESCs. E2 affects angiogenesis and growth in the human endometrium via a synergistic pathway
involving both VEGF and SDF-1. With regard to SDF-1, steroid hormones rather than hypoxia may be the main regulator for its secretion
in ESCs. These results indicate that SDF-1 plays a more important role
for endometrial proliferation than repair of the endometrium during
the perimenstrual period. We have previously described SDF-1 as a
candidate factor involved in the epithelial-stromal interaction of the
527
Figure 3 The dose-dependent effect of CoCl2 on VEGF (A) and
SDF-1 (B) secretion from ESCs. ESCs were grown for 48 h in
medium containing either control or CoCl2 (0.1– 100 mM). ELISA
measured VEGF and SDF-1 concentrations in the culture medium.
Results are representative data of four experiments with different
cell preparations and each value represents mean + SEM.
*P , 0.05, **P , 0.01 versus control.
human endometrium and concluded that SDF-1 might be actively
involved in its proliferation (Tsutsumi et al., 2011).
The present study showed that hypoxia-induced VEGF secretion
was significantly attenuated by echinomycin. Echinomycin is a
sequence-specific DNA-binding agent that binds to the HRE site
within the promoters of HIF-1a target genes and selectively inhibits
HIF-1a binding activity (Kong et al., 2005). We confirmed that the
inhibition of VEGF production by echinomycin did not affect HIF-1a
protein levels and cell viability. These results indicate that echinomycin
can inhibit VEGF expression, which is a key regulator of angiogenesis
and vascular function in the human endometrium, without changing
the HIF-1a protein level and causing cell toxicity. These results
demonstrate that echinomycin may be a potential therapeutic agent
for HIF-1a-dependent disorders and normal function. In addition,
the inhibition of HIF-1a function by echinomycin has been shown to
block ovulation in mice, and echinomycin reduced the expression of
VEGF, a key factor controlling angiogenesis during ovulation (Kim
et al., 2009). Furthermore, phases I and II cancer therapeutic trials
of echinomycin have been based on observations that echinomycin
528
Tsuzuki et al.
Figure 4 (A) Western blot analysis for HIF-1a protein expression.
Total cell lysates obtained from ESCs cultured with either control or
CoCl2 (100 mM) for 2, 6, 24 and 48 h were loaded onto a 7.5%
sodium dodecyl sulfate– polyacrylamide gel. (B) The protein levels
were quantified using ImageJ. Columns and vertical bars represent
the mean + SEM for combined data of three separate experiments
with different cell preparations. *P , 0.05, **P , 0.01 versus control.
exhibits potent anti-proliferative effects on several tumor-derived cell
lines (Foster et al., 1985; Gradishar et al., 1995).
What is the mechanism underlying the inhibition of SDF-1 production under hypoxic conditions? We showed that hypoxia attenuated
SDF-1 production in ESCs but echinomycin has no effect on this
suppression. Hypoxia is known to induce the activation of transcription factors, including HIF-1a, activator protein 1 (AP-1) and nuclear
factor (NF)-kB (Lukiw et al., 2003; Martin et al., 2004). As previously
shown, echinomycin specifically inhibited HIF-1a-dependent, but not
AP-1- or NF-kB-dependent, DNA-binding activity (Kong et al.,
2005). Therefore, we speculate that the inhibitory effect on SDF-1
production by hypoxia is caused by the action of transcription factor
AP-1 or NF-kB. However, the precise mechanisms by which
hypoxia acts to inhibit SDF-1 production are not fully understood.
We revealed that hypoxia significantly increased the VEGF production levels and attenuated SDF-1 production in human ESCs. These
results are in agreement with earlier studies describing that the
VEGF mRNA level was increased and the SDF-1 mRNA level was
decreased in culture cells of hypoxic rat inner ear (Gross et al.,
2009). This induction of ESC-derived VEGF production may contribute to the early angiogenic processes associated with postmenstrual
Figure 5 Effects of CoCl2 and/or echinomycin on VEGF (A) and
SDF-1 (B) secretion from ESCs. ESCs were grown for 48 h in
medium containing with either CoCl2 (100 mM) and/or echinomycin
(0.1– 100 nM, a small-molecule inhibitor of HIF-1a activity). ELISA
measured VEGF and SDF-1 concentrations in the culture medium.
The effect of CoCl2 alone is assigned a potency of 100%. Columns
and vertical bars represent the mean + SEM for combined data of
three separate experiments. Values are significantly different
(*P , 0.01 versus CoCl2 alone). (C) LDH assay measured dose – response cell viability treated with different concentrations of CoCl2
and echinomycin. Cell culture medium treated with 5% Tween 20
was used as positive control. Results are representative data of
three experiments with different cell preparations and each value
represents mean + SEM. *P , 0.01 versus vehicle.
regeneration of endometrium (Fan et al., 2008). The decrease of
SDF-1 production may be involved in the mobilization of local endothelial progenitor or stem cells in the regenerating endometrium
(Semerad et al., 2005; Gross et al., 2009).
In summary, we showed that hypoxia simultaneously acts to
increase VEGF via HIF-1a and to decrease SDF-1 in a
HIF-1a-independent manner in ESCs. These data indicate a potential
mechanism for the action of hypoxic conditions that could influence
angiogenesis of the human endometrium. Moreover, echinomycin
529
Hypoxia affects the angiogenic factors
Funding
This work was supported in part by grants-in-aid 23592425 and
23592426 for Scientific Research from the Ministry of Education,
Science and Culture, Japan.
Conflict of interest
None declared.
References
Figure 6 (A) Western blot analysis for HIF-1a protein expression.
Total cell lysates were obtained from ESCs treated with control or
the presence or absence of CoCl2 (100 mM) and echinomycin
(0.1– 100 nM) for 6 h. (B) Columns and vertical bars represent the
mean + SEM for combined data of three separate experiments.
*P , 0.01 versus control.
can inhibit VEGF production in ESCs, without affecting the HIF-1a
protein level, SDF-1 production and cell toxicity. These findings
suggest a unique therapeutic potential for echinomycin as an inhibitor
of HIF-1a activation. The knowledge of angiogenic factors in the endometrium will have an impact on the development of strategies for inhibiting or augmenting the growth of blood vessels in normal and
pathologic human reproductive processes. In addition, understanding
the role of O2 tension on the regulation of these factors might help
clarify the mechanism of regulation of physiologic angiogenesis in
normal tissues.
Acknowledgements
The authors thank Ms Rika Okamoto for excellent technical
assistance.
Authors’ roles
All authors have met the criteria for justification of authorship. They
have all substantially contributed to the conception/design/acquisition
of data/analysis and interpretation of data; drafted or revised the
article for important intellectual content; participated in the finalization
of the manuscript and approved the final draft.
Aberdeen GW, Wiegand SJ, Bonagura TW Jr, Pepe GJ, Albrecht ED.
Vascular endothelial growth factor mediates the estrogen-induced
breakdown of tight junctions between and increase in proliferation of
microvessel endothelial cells in the baboon endometrium.
Endocrinology 2008;149:6076 – 6083.
Ardyanto TD, Osaki M, Tokuyasu N, Nagahama Y, Ito H. CoCl2-induced
HIF-1alpha expression correlates with proliferation and apoptosis in
MKN-1 cells: a possible role for the PI3K/Akt pathway. Int J Oncol
2006;29:549 – 555.
Bachelder RE, Wendt MA, Mercurio AM. Vascular endothelial growth
factor promotes breast carcinoma invasion in an autocrine manner by
regulating the chemokine receptor CXCR4. Cancer Res 2002;
62:7203– 7206.
Burger JA, Kipps TJ. CXCR4: a key receptor in the crosstalk between
tumor cells and their microenvironment. Blood 2006;107:1761 –1767.
Ceradini DJ, Gurtner GC. Homing to hypoxia: HIF-1 as a mediator of
progenitor cell recruitment to injured tissue. Trends Cardiovasc Med
2005;15:57 – 63.
Cho J, Kim D, Lee S, Lee Y. Cobalt chloride-induced estrogen receptor
alpha down-regulation involves hypoxia-inducible factor-1alpha in
MCF-7 human breast cancer cells. Mol Endocrinol 2005;19:1191– 1199.
Critchley HO, Osei J, Henderson TA, Boswell L, Sales KJ, Jabbour HN,
Hirani N. Hypoxia-inducible factor-1alpha expression in human
endometrium and its regulation by prostaglandin E-series prostanoid
receptor 2 (EP2). Endocrinology 2006;147:744 – 753.
Dominguez F, Galan A, Martin JJ, Remohi J, Pellicer A, Simon C. Hormonal
and embryonic regulation of chemokine receptors CXCR1, CXCR4,
CCR5 and CCR2B in the human endometrium and the human
blastocyst. Mol Hum Reprod 2003;9:189 – 198.
Dong X, Wang YS, Dou GR, Hou HY, Shi YY, Zhang R, Ma K, Wu L,
Yao LB, Cai Y et al. Influence of Dll4 via HIF-1alpha-VEGF signaling
on the angiogenesis of choroidal neovascularization under hypoxic
conditions. PLoS One 2011;6:e18481.
Fan X, Krieg S, Kuo CJ, Wiegand SJ, Rabinovitch M, Druzin ML,
Brenner RM, Giudice LC, Nayak NR. VEGF blockade inhibits
angiogenesis and reepithelialization of endometrium. FASEB J 2008;
22:3571– 3580.
Foster BJ, Clagett-Carr K, Shoemaker DD, Suffness M, Plowman J,
Trissel LA, Grieshaber CK. Leyland-Jones B. Echinomycin: the first
bifunctional intercalating agent in clinical trials. Invest New Drugs 1985;
3:403 – 410.
Gradishar WJ, Vogelzang NJ, Kilton LJ, Leibach SJ, Rademaker AW,
French S, Benson AB III. A phase II clinical trial of echinomycin in
metastatic soft tissue sarcoma. An Illinois Cancer Center Study. Invest
New Drugs 1995;13:171 – 174.
Gross J, Moller R, Amarjargal N, Machulik A, Fuchs J, Ungethum U,
Kuban RJ, Henke W, Haupt H, Mazurek B. Expression of
erythropoietin and angiogenic growth factors following inner ear injury
of newborn rats. Prague Med Rep 2009;110:310 – 331.
530
Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh M, Salic A, Asara JM,
Lane WS, Kaelin WG Jr. HIF alpha targeted for VHL-mediated
destruction by proline hydroxylation: implications for O2 sensing.
Science 2001;292:464 – 468.
Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, Gaskell SJ,
Kriegsheim A, Hebestreit HF, Mukherji M, Schofield CJ et al. Targeting
of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by
O2-regulated prolyl hydroxylation. Science 2001;292:468 – 472.
Kim J, Bagchi IC, Bagchi MK. Signaling by hypoxia-inducible factors is critical
for ovulation in mice. Endocrinology 2009;150:3392 – 3400.
Kong D, Park EJ, Stephen AG, Calvani M, Cardellina JH, Monks A,
Fisher RJ, Shoemaker RH, Melillo G. Echinomycin, a small-molecule
inhibitor of hypoxia-inducible factor-1 DNA-binding activity. Cancer
Res 2005;65:9047– 9055.
Lukiw WJ, Ottlecz A, Lambrou G, Grueninger M, Finley J, Thompson HW,
Bazan NG. Coordinate activation of HIF-1 and NF-kappaB DNA binding
and COX-2 and VEGF expression in retinal cells by hypoxia. Invest
Ophthalmol Vis Sci 2003;44:4163 – 4170.
Martin G, Andriamanalijaona R, Grassel S, Dreier R, Mathy-Hartert M,
Bogdanowicz P, Boumediene K, Henrotin Y, Bruckner P, Pujol JP.
Effect of hypoxia and reoxygenation on gene expression and response
to interleukin-1 in cultured articular chondrocytes. Arthritis Rheum
2004;50:3549 – 3560.
Martin SK, Diamond P, Williams SA, To LB, Peet DJ, Fujii N, Gronthos S,
Harris AL, Zannettino AC. Hypoxia-inducible factor-2 is a novel
regulator of aberrant CXCL12 expression in multiple myeloma plasma
cells. Haematologica 2010;95:776– 784.
Nakamoto T, Okada H, Nakajima T, Ikuta A, Yasuda K, Kanzaki H.
Progesterone induces the fibulin-1 expression in human endometrial
stromal cells. Hum Reprod 2005;20:1447 – 1455.
Nishigaki A, Okada H, Okamoto R, Sugiyama S, Miyazaki K, Yasuda K,
Kanzaki H. Concentrations of stromal cell-derived factor-1 and
vascular endothelial growth factor in relation to the diameter of
human follicles. Fertil Steril 2010;95:742– 746.
Okada H, Nakajima T, Yoshimura T, Yasuda K, Kanzaki H. The inhibitory
effect of dienogest, a synthetic steroid, on the growth of human
endometrial stromal cells in vitro. Mol Hum Reprod 2001;7:341 – 347.
Okada H, Nie G, Salamonsen LA. Requirement for proprotein convertase
5/6 during decidualization of human endometrial stromal cells in vitro.
J Clin Endocrinol Metab 2005;90:1028 – 1034.
Okada H, Tsutsumi A, Imai M, Nakajima T, Yasuda K, Kanzaki H. Estrogen
and selective estrogen receptor modulators regulate vascular
endothelial growth factor and soluble vascular endothelial growth
factor receptor 1 in human endometrial stromal cells. Fertil Steril
2009;93:2680 – 2686.
Okada H, Okamoto R, Tsuzuki T, Tsuji S, Yasuda K, Kanzaki H. Progestins
inhibit estradiol-induced vascular endothelial growth factor and stromal
cell-derived factor 1 in human endometrial stromal cells. Fertil Steril
2011;96:786 – 791.
Popovici RM, Irwin JC, Giaccia AJ, Giudice LC. Hypoxia and cAMP
stimulate vascular endothelial growth factor (VEGF) in human
Tsuzuki et al.
endometrial stromal cells: potential relevance to menstruation and
endometrial regeneration. J Clin Endocrinol Metab 1999;84:2245– 2248.
Ruiz A, Salvo VA, Ruiz LA, Baez P, Garcia M, Flores I. Basal and steroid
hormone-regulated expression of CXCR4 in human endometrium and
endometriosis. Reprod Sci 2010;17:894 – 903.
Salamonsen LA. Tissue injury and repair in the female human reproductive
tract. Reproduction 2003;125:301 – 311.
Salcedo R, Wasserman K, Young HA, Grimm MC, Howard OM,
Anver MR, Kleinman HK, Murphy WJ, Oppenheim JJ. Vascular
endothelial growth factor and basic fibroblast growth factor induce
expression of CXCR4 on human endothelial cells: in vivo
neovascularization induced by stromal-derived factor-1alpha. Am J
Pathol 1999;154:1125– 1135.
Schioppa T, Uranchimeg B, Saccani A, Biswas SK, Doni A, Rapisarda A,
Bernasconi S, Saccani S, Nebuloni M, Vago L et al. Regulation of the
chemokine receptor CXCR4 by hypoxia. J Exp Med 2003;
198:1391– 1402.
Semenza GL. Oxygen sensing, homeostasis, and disease. N Engl J Med
2011;365:537 – 547.
Semerad CL, Christopher MJ, Liu F, Short B, Simmons PJ, Winkler I,
Levesque JP, Chappel J, Ross FP, Link DC. G-CSF potently inhibits
osteoblast activity and CXCL12 mRNA expression in the bone
marrow. Blood 2005;106:3020 – 3027.
Sharkey AM, Day K, McPherson A, Malik S, Licence D, Smith SK,
Charnock-Jones DS. Vascular endothelial growth factor expression in
human endometrium is regulated by hypoxia. J Clin Endocrinol Metab
2000;85:402 – 409.
Sugino N, Kashida S, Karube-Harada A, Takiguchi S, Kato H. Expression of
vascular endothelial growth factor (VEGF) and its receptors in human
endometrium throughout the menstrual cycle and in early pregnancy.
Reproduction 2002;123:379 – 387.
Szablowska-Gadomska I, Zayat V, Buzanska L. Influence of low oxygen
tensions on expression of pluripotency genes in stem cells. Acta
Neurobiol Exp (Wars) 2011;71:86– 93.
Takenaga K. Angiogenic signaling aberrantly induced by tumor hypoxia.
Front Biosci 2011;16:31 –48.
Tsutsumi A, Okada H, Nakamoto T, Okamoto R, Yasuda K, Kanzaki H.
Estrogen induces stromal cell-derived factor 1 (SDF-1/CXCL12)
production in human endometrial stromal cells: a possible role of
endometrial epithelial cell growth. Fertil Steril 2011;95:444 – 447.
Wu X, Jin LP, Yuan MM, Zhu Y, Wang MY, Li DJ. Human first-trimester
trophoblast cells recruit CD56brightCD16- NK cells into decidua by
way of expressing and secreting of CXCL12/stromal cell-derived
factor 1. J Immunol 2005;175:61 – 68.
Yamakawa M, Liu LX, Date T, Belanger AJ, Vincent KA, Akita GY,
Kuriyama T, Cheng SH, Gregory RJ, Jiang C. Hypoxia-inducible
factor-1 mediates activation of cultured vascular endothelial cells by
inducing multiple angiogenic factors. Circ Res 2003;93:664– 673.
Yoshie M, Miyajima E, Kyo S, Tamura K. Stathmin, a microtubule regulatory
protein, is associated with hypoxia-inducible factor-1alpha levels in human
endometrial and endothelial cells. Endocrinology 2009;150:2413– 2418.