| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
Reproductive Molecular Research Group, Department of Obstetrics and Gynecology, University of Cambridge, Rosie Hospital, Cambridge, United Kingdom CB2 2SW
Address all correspondence and requests for reprints to: Dr. Andrew Sharkey, Department of Obstetrics and Gynecology, University of Cambridge, Box 223, Rosie Maternity Hospital, Robinson Way, Cambridge, United Kingdom CB2 2SW. E-mail: ams{at}mole.bio.cam.ac.uk
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Angiogenesis involves the degradation of extracellular matrix and the migration, proliferation, and formation of tubes by endothelial cells (3). Vascular endothelial growth factor (VEGF) is a secreted protein that stimulates endothelial cell proliferation and migration; it also increases vascular permeability (4). Two receptors bind VEGF with high affinity: the Fms-like tyrosine kinase (Flt-1) and the kinase insert domain receptor KDR (5, 6). Disruption of the gene encoding VEGF or its receptors in mice results in failure of endothelial cell differentiation and angiogenesis (7, 8, 9, 10). Injection of VEGF induces blood vessel development in vivo, and these actions are inhibited by antibodies to VEGF or soluble Flt-1 receptor (11, 12, 13, 14). Thus, VEGF plays a pivotal role in both normal and pathological angiogenesis (15, 16).
Alternative splicing results in five forms of the VEGF protein, with 206-, 189-, 165-, 145-, and 121-amino acid residues (17, 18). These isoforms differ in biological activity; VEGF165 and VEGF121 are secreted freely, whereas VEGF189 and VEGF206 are predominantly cell associated (17). Four other closely related genes have been described, including placental growth factor, VEGF-B, VEGF-C, and VEGF-D, giving rise to a gene family, each member of which is involved in regulating blood vessel development and function (4, 19, 20).
Hormonal regulation of the level and site of expression of VEGF in the uterus has been demonstrated in several species. In ovariectomized rats, estrogen and progesterone increase uterine VEGF messenger ribonucleic acid (mRNA) levels within 2 h of administration (21, 22). In human endometrium, VEGF mRNA levels increase during the secretory phase of the cycle (23, 24). Culture of stromal cells from human endometrium in the presence of estradiol and medroxyprogesterone acetate results in a 4-fold increase in VEGF mRNA levels, consistent with the in vivo data (23). In situ hybridization and immunocytochemical studies show that VEGF mRNA and immunoreactivity increase in glandular epithelium during the luteal phase, with diffuse and variable staining of stromal cells throughout the cycle (18, 23, 24, 25). These data suggest that the level of VEGF expression in both glandular and stromal cells from endometrium is regulated by steroids. However, in situ hybridization with a probe specific for VEGF mRNA shows intense hybridization in menstrual endometrium, when steroid levels are low, indicating that additional factors regulate VEGF levels in endometrium (18).
The purpose of this study was to investigate the role of hypoxia as a local regulator of VEGF expression in human endometrium. Hypoxia is an important regulator of VEGF mRNA levels in many tissues, including granulosa cells, glioblastoma tumor cells, and the developing retina (15, 26, 27). As menstruation is characterized by distal ischemia in endometrium, the effects of hypoxia on VEGF secretion and mRNA levels were examined in primary cultures of stromal and epithelial cells from endometrium. The relative contributions of steroid hormones and hypoxia to the regulation of VEGF expression in endometrium were determined.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Endometrial tissues were collected from 20 patients after dilatation and curettage or hysterectomy performed for benign gynecological conditions. The majority of patients were undergoing investigation of subjective menorrhagia, and none was using steroidal contraception or intrauterine contraceptive devices. It has previously been shown that over 50% of women complaining of menorrhagia in fact have blood loss values within the normal range. Tissue from most of these women should therefore have been normal. All biopsies used experimentally were histologically normal. Primary endometrial cell cultures were established from the fresh endometrial biopsies, and duplicate specimens were fixed in formalin and processed for in situ hybridization. Informed consent was obtained from the patients, and the study was approved by the ethical committees of Addenbrooke’s Hospital National Health Service Trust (Cambridge, UK).
Human endometrial cell culture
All tissue culture and laboratory reagents were purchased from Sigma (Poole, UK) unless otherwise stated. After biopsy, glands and stroma were separated by digestion in Hanks’ Buffered Saline with collagenase (1 mg/mL) and deoxyribonuclease (0.5 mg/mL) and filtration through a fine 40-µm pore size nylon mesh (Becton Dickinson, Plymouth, UK). Glands retained on the sieve were further digested with collagenase for 2 h at 37 C, washed twice in phosphate-buffered saline, and plated in 48-well plates at 1.2 x 105 cells/well in 0.5 mL phenol-red free DMEM-Ham’s F-12 containing penicillin (50 IU/mL), streptomycin (50 µg/mL), L-glutamine (2 mmol/L), 2% FCS, and insulin (10 µg/mL). Stromal cells were initially plated in 25-cm2 flasks in phenol red-free DMEM-Ham’s F-12 as described above, but with FCS at 10% and without insulin. All serum was purchased charcoal stripped to remove steroids and growth factors. To determine VEGF secretion by stromal cells, primary cells were passaged into 48-well plates, grown to confluence, and then used for hypoxia or steroid experiments.
Growth of cells under hypoxic or normoxic conditions
For experiments involving growth of the cells under hypoxic conditions, cells were plated in 48-well plates in triplicate and cultured for 24 h as described above. For hypoxic culture, a jet of 95% nitrogen-5% CO2 was used to flush out each plate for 2 min, and the plates were then sealed in an airtight container and incubated in the same gaseous mixture for 24 h. Before harvest, the partial pressure of O2 in the medium was measured using a blood gas analyzer (Instrumentation Laboratory Ltd., Warrington, UK). A duplicate set of control cells was cultured in 5% CO2 under normal atmospheric oxygen tension. After culture under hypoxic or normoxic conditions for 24 h, the supernatants were harvested and stored at -20 C for analysis of VEGF levels by enzyme-linked immunosorbent assay (ELISA). The purity of the glandular and stromal preparations was more than 90%, as judged by positive cellular staining for cytokeratin or vimentin, respectively, on cytospin preparations of the same cells. Staining for cytokeratin and vimentin used antibodies MNF116 and 3B4 (DAKO Corp., High Wycombe, UK) as described previously (28).
Quantitation of VEGF mRNA in isolated endometrial epithelial and stromal cells
Total RNA was isolated from 48-well plates of glandular epithelium cells grown identically as those used to compare VEGF secretion in normoxic and hypoxic conditions. After removal of the culture supernatant for ELISA, total RNA was isolated using the RNEasy kit (QIAGEN, Crawley, UK). The VEGF mRNA content in the total RNA from each well was measured using a fluorescent nested RT-PCR method described by us previously (29, 30). In brief, this involves the addition to each RNA sample of a known amount of a complementary RNA construct that is amplified by the same primers as the target VEGF mRNA. After complementary DNA (cDNA) synthesis using 1/10th of the RNA from each well, the construct and the 4 VEGF cDNAs were amplified with primers I and J using the following profile: 95 C for 1 min, then 95, 60, and 72 C each for 30 s for 20 cycles, followed by 3 min at 72 C to ensure complete extension. From the 30 µL of first round product, 1.5 µL were diluted into 30 µL PCR mix containing the nested primers carboxyfluorescein and H. Four serial dilutions of this stock were made (1:3 each time), producing 5 different dilutions of the first round product, each of 20 µL. These were then amplified for an additional 20 cycles as described above, except the annealing temperature of each cycle was 64 C. Four microliters of loading buffer [15% Ficoll 400-DL (Pharmacia Biotech, Uppsala, Sweden), 4.15 mg/mL blue dextran (Sigma, D-5751), 89 mmol/L Tris-borate, and 1 mmol/L ethylenediamine tetraacetate] were added to each reaction tube. Two microliters of the fluorescently labeled products were loaded onto a 0.4-mm thick 6% nondenaturing polyacrylamide gel and electrophoresed using an ABI 373A DNA sequencer equipped with Genescan 1.1 software (PE Applied Biosystems, Warrington, UK). The sequences of the primers were described previously (28). For fluorescent detection, addition of a carboxyfluorescein-labeled phosphoramidite was made to the 5'-end of primer C. Five peaks were detected in each sample, corresponding to the cDNAs of the standard construct, VEGF121, VEGF145, VEGF165, and VEGF189. Log plots of the peak areas for the serially diluted samples gave parallel curves for the standard and the four VEGF cDNA species. The ratio between each VEGF cDNA species and the standard was calculated, and the amount of each mRNA species in a culture well was determined as femtograms per well.
Quantitation of VEGF mRNA in endometrial stromal cells
Endometrial stromal cells were passaged once into 75-cm2 flasks, grown to confluence, and then cultured under normoxic or hypoxic conditions as described above. Total RNA was extracted using a modification of the single step guanidine thiocyanate-phenol-chloroform extraction method (18). VEGF mRNA levels were quantified by quantitative RT-PCR as described above, except that the results were expressed as femtograms of VEGF mRNA per µg of input total RNA. This was possible because more RNA was obtained from stromal than epithelial cells cultured in vitro, so the amount of input RNA from stromal cells used for cDNA synthesis could be measured.
ELISAs
Levels of VEGF in culture supernatants were determined in duplicate samples by enzyme immunoassay using a commercially available pair of antibodies designed for this purpose (R&D Systems, Oxford, UK). The ELISA was performed according to the manufacturer’s instructions. The limit of sensitivity of the assay was 20 pg/mL. The intra- and interassay coefficients of variation were 4.5% and 6.7%, respectively.
Statistical analysis
For comparison of VEGF secretion from endometrial cells cultured under hypoxic conditions with that in control cultures, the results obtained from a single patient’s cells for each treatment were compared using Student’s t test. VEGF secretion from glandular epithelium isolated from secretory phase endometrium and that from proliferative phase endometrium were compared using the nonparametric Mann-Whitney U test. Statistical significance was accepted at P < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Primary cultures of stromal and epithelial cells were cultured
separately under normoxic and hypoxic conditions for 24 h. Culture
supernatants were analyzed for VEGF levels by ELISA. The
pO2 in the culture supernatant of cells grown in
normal oxygen was 19–21 kPa, whereas in the hypoxic cultures it was
8–11 kPa. Results from four separate experiments with stromal cells
and eight with glandular epithelium are shown in Fig. 1
. In normal culture, stromal cells
secreted a mean level of VEGF of 41 pg/105
cells/24 h (SEM, ±5.1). After growth in hypoxic conditions
for 24 h, all stromal cell samples showed increased secretion of
VEGF, with mean VEGF secretion of 158 pg/105
cells/24 h (SEM, ±29). The increase in VEGF between the
hypoxic and normal stromal cells in each experiment was highly
significant (P < 0.02) and ranged from 2.1- to
6.1-fold, with a mean increase of 3.6-fold. The mean level of VEGF
secretion from epithelial cells isolated from proliferative phase
endometrium and grown under normal conditions was 138
pg/105 cells·24 h (SEM,
±29.4), and that from cells isolated from secretory endometrium was
401 pg/105 cells·24 h
(SEM, ±33.8). Hypoxia increased this to 403
pg/105 cells·24 h (SEM,
±82) for proliferative cells and 1813 pg/105
cells·24 h (SEM, ±669) for secretory cells.
The average increases after hypoxia were 3.4- and 4.4-fold,
respectively, and were highly significant for all experiments
(P < 0.02). The basal secretion of VEGF by glandular
epithelial cells harvested from secretory endometrium and cultured
under normoxic conditions was significantly higher than that from cells
from proliferative endometrium (P = 0.036;
asterisked in Fig. 1). Duplicate experiments using
epithelial cells carried out in serum-free medium yielded similar
results (data not shown). These increases are not due to necrosis or
cell death, because after 24 h of hypoxia treatment both stromal
and epithelial cells remain viable, as judged by trypan blue
exclusion.
|
To measure changes in VEGF mRNA levels in glandular epithelial
cells in response to hypoxia, a quantitative RT-PCR method was used.
This was necessary because relatively few gland cells are obtained
after primary culture. VEGF mRNA levels were measured in total RNA
extracted from glandular epithelium cells that were cultured under
normoxic and hypoxic conditions. Cells from three separate patients
were used. A known amount of a complementary RNA construct was added to
the RNA from each well and subjected to RT-PCR using primers for VEGF.
After RT-PCR, four products were detected of 176, 308, 380, and 414 bp,
corresponding to the mRNA species encoding
VEGF121, VEGF165,
VEGF189, and the artificial construct. A dilution
series of the first round product was used for the second round to
determine whether linear amplification was occurring (Fig. 2A). Although a signal for
VEGF145 was present, this was at the limit of
detection and could not be reliably measured under the conditions used.
Figure 2B shows an example of the log plot of the peak intensity for
the construct and each VEGF mRNA species at different dilutions after
electrophoresis of second round PCR products using an ABI373A
sequencer. The linear relationship between the amount of added control
template and peak intensity indicates that linear amplification is
occurring. The mean ratio between the added construct and each VEGF
splice variant allows the amount of each product to be determined. The
results are shown in Table 1. Under
normal conditions the total amounts of VEGF mRNA for each patient were
184, 439, and 162 fg/well, whereas after hypoxia these values were 581,
1724, and 430 fg/well, respectively (Table 1). This represents
increases in total VEGF mRNA levels of 3.2-, 3.9-, and 2.7-fold after
hypoxia. The RT-PCR approach allows the accurate quantitation in
endometrial gland cells of the relative levels of each of the VEGF
splice variants. The amount of VEGF145 was the
lowest, followed by VEGF189, with
VEGF165 and VEGF121 being
the most abundant. The ratios of the splice variants were not altered
significantly in each patient after hypoxia.
|
|
. Under normal conditions the total
amounts of VEGF mRNA were 38.8, 127.9, and 63.2 fg/µg total RNA,
whereas after hypoxia these values were 73.1, 275.1, and 81.4 fg/µg
total RNA. This represents an average increase in VEGF mRNA of 1.8-fold
in response to hypoxia for the three experiments. In stromal cells the
VEGF165 and VEGF189 mRNA
species were the most abundant, and the relative ratios of each splice
variant did not alter after hypoxia.
|
VEGF secretion by endometrial cells has previously been reported
to be regulated by steroids. We therefore examined whether treatment
with estrogen or progesterone altered VEGF secretion in response to
hypoxia. Endometrial stromal (n = 5) or epithelial (n = 5)
cells were cultured as before in 48-well plates under hypoxic or
normoxic conditions in the presence or the absence of
10-10 mol/L estrogen or
10-8 mol/L progesterone. VEGF secretion was
measured after 24 h by ELISA. The results for each treatment are
expressed as VEGF secretion relative to the untreated cells from the
same patient. As before, a significant increase in VEGF secretion by
both gland and stromal cells in response to hypoxia was detected
(P < 0.001 for both cell types; Fig. 3, A and B). This response was unaffected
by the presence of either steroid. No significant change in VEGF
secretion was detected after treatment with either steroid alone.
Although the baseline VEGF secretion differed in the untreated cells
from different patients, similar percent increases in VEGF secretion in
response to hypoxia were obtained from repeated experiments.
|
In situ hybridization was used to examine the
localization of VEGF mRNA in menstrual endometrium. This was compared
with VEGF expression in the proliferative and secretory phases of the
cycle. Strong hybridization of the VEGF antisense probe was seen in
menstrual endometrium and was localized to both glandular and stromal
compartments (Fig. 4, G–I). This is in
contrast to the proliferative phase, when expression of VEGF was
primarily stromal, with negligible hybridization to glands (Fig. 4, A–C). During the secretory phase VEGF mRNA expression was primarily
seen in the glandular epithelium, with little stromal expression (Fig. 4, D–F). Thus, during menstruation, VEGF expression is simultaneously
expressed in both compartments of the endometrium.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Hypoxia is an important stimulus for new blood vessel formation (15, 31). The effect of hypoxic stimulation of angiogenesis is accompanied by increased levels of VEGF mRNA (4, 15). This occurs both by up-regulation of VEGF transcription and by increased VEGF mRNA stability (32). Menstruation is characterized by blood vessel constriction and distal ischemia, and hypoxia is therefore likely to be a physiologically relevant regulator of endometrial repair. A fluorescent quantitative RT-PCR method was used to measure VEGF mRNA levels in endometrial epithelial and stromal cells cultured separately under normoxic and hypoxic conditions (29, 30). This approach allowed quantitation of both total VEGF mRNA and individual splice variants. After hypoxia, total VEGF mRNA increased by an average of 3.2-fold in epithelial cells and 1.8-fold in stromal cells. The increases in VEGF mRNA levels are comparable to those in VEGF protein secretion in the same cultures. The increased secretion in response to hypoxia is therefore largely due to increased steady state levels of VEGF mRNA. The extent of up-regulation of VEGF mRNA in endometrial cells by hypoxia is similar to that reported in a number of in vivo and in vitro studies using different tissues and cell lines (33, 34). For example, the human choriocarcinoma cell line JEG-3 showed a 4-fold increase in VEGF secretion after culture under hypoxic conditions. This was accompanied by a similar increase in VEGF mRNA levels (34). The ability to quantitate the levels of each splice variant of VEGF allowed us to identify VEGF165 and VEGF121 as the most abundant variants in epithelial cells. Levels of VEGF145 were at the lower limit of detection. These ratios confirm those reported in endometrial stromal and epithelial cells using nonquantitative RT-PCR (24, 35). The ratio of the splice variants in both epithelial and stromal cells was not altered by hypoxia, with all increasing to the same extent.
Steroids and hypoxia
There is considerable evidence that steroids regulate VEGF expression in the stromal and epithelial compartments of the endometrium. Culture of stromal cells with estradiol or with estradiol and progesterone together increased VEGF mRNA levels 3.1- and 4.7-fold in serum-free conditions (23). However, the corresponding changes in VEGF secretion were modest (0.46- and 0.18-fold, respectively). We did not find a comparable increase in VEGF secretion by stromal cells in response to estradiol, although our experiments were carried out in charcoal-stripped serum, which may explain the different result. In vivo, several studies show that VEGF mRNA levels are increased during the secretory phase, indicating up-regulation by progesterone (18, 22, 23, 24). Administration of exogenous steroids to castrate cynomolgus monkeys confirmed that VEGF expression in endometrium is regulated by the steroidal environment (36). In view of this evidence for steroidal regulation, we investigated the effect of estrogen and progesterone on the response of both glandular and stromal cells to hypoxia. The presence of steroids had no effect on the increase in VEGF secretion in response to hypoxia, indicating that the steroidal and hypoxic responses are independent, as has been found in human breast carcinoma cell lines (37).
In situ hybridization to localize VEGF mRNA through the cycle indicates that during the proliferative phase, VEGF mRNA is largely confined to the stroma, with little glandular expression. This accords with the in vitro up-regulation of VEGF mRNA by estrogen in stromal cells (23). In the secretory phase, in situ hybridization showed decreased stromal VEGF mRNA, but expression in the glands was greatly increased, presumably under the influence of progesterone. In support of this, we found that secretion of VEGF from secretory epithelial cells was 3-fold higher than that in epithelial cells from proliferative endometrium. In contrast, during menstruation there was strong hybridization of the VEGF probe to both glands and stroma, indicating simultaneous expression of VEGF mRNA in both compartments. This is at a time when steroid levels are low, and we postulate that it is due to up-regulation of VEGF in response to tissue ischemia. High levels of VEGF secretion at this time may be important in the process of endometrial repair and angiogenesis that follows menstruation.
The proteins encoded by the different VEGF splice variants appear to have different biological activities. VEGF121 has been shown to be freely secreted, whereas VEGF189 (and, to a lesser degree, VEGF165) bind to extracellular matrix (ECM) (17). Human endometrial cells, therefore, synthesize VEGF isoforms that are freely secreted and those that are sequestered in the ECM. Large amounts of these ECM-bound forms released at menstruation may serve as a depot and be made available through proteolysis of ECM during subsequent remodeling of the endometrium (38).
Mechanism of VEGF regulation by hypoxia
The increased VEGF mRNA levels reported in a number of cell lines in response to hypoxia are due to both up-regulation of VEGF transcription and increased VEGF mRNA stability (32, 34). The mechanisms by which this occurs are now being clarified. The transcription factor hypoxia-inducible factor-1 up-regulates a number of genes in the cellular response to hypoxia, including the glycolytic enzymes and erythropoietin. Hypoxia-inducible factor-1 binds to the VEGF promoter, activating transcription (39). At the same time, specific sequences have been identified in the 3'-noncoding region of the mRNA, which render it unstable and susceptible to rapid degradation. This breakdown can be blocked by actinomycin D or culture in hypoxic conditions (32). These researchers propose that hypoxic stress induces proteins that bind to these instability sequences and prevent rapid degradation of the VEGF mRNA, leading to increased steady state levels. It will be interesting to examine whether similar mechanisms operate in the uterine endometrium at menstruation to regulate VEGF mRNA levels.
The finding that hypoxia up-regulates VEGF in endometrium may be relevant to understanding the etiology of endometriosis. This condition probably arises by the deposition of fragments of endometrium in the peritoneal cavity by retrograde menstruation (40). However, little is known of the mechanisms that allow such tissue fragments to attach and develop a new vascular supply, steps that are crucial in the establishment of ectopic explants. The finding that VEGF is strongly up-regulated in endometrium by hypoxia suggests that endometrial fragments shed at menstruation and exposed to hypoxic stress could settle in the peritoneal cavity, releasing large amounts of VEGF. This, in turn, would induce local angiogenesis and aid revascularization of the endometrium at the ectopic site. At menstruation, the endometrium also releases large amounts of metalloproteinases (MMPs), which are believed to participate in the process of remodeling and repair in normal endometrium (41). However, in the peritoneum, this local MMP release may allow the refluxed endometrial fragments to breach the mesothelium and establish a direct vascular connection with peritoneal blood vessels. The activation of revascularization and repair systems, as represented by VEGF and MMPs, for normal endometrial repair after menstruation may therefore also allow implantation in the peritoneum. Thus, implantation at ectopic sites may be a consequence of the normal biological response of the endometrium to the process of menstruation.
This study has shown that hypoxia is a potent regulator of VEGF secretion from both glandular and stromal cells of human endometrium. In both cases the mechanism involves increases in the steady state levels of VEGF mRNA. Hypoxia is a physiologically relevant regulator, as distal ischemia occurs in the endometrium due to constriction of the spiral arterioles at menstruation. The up-regulation of VEGF by hypoxia may play an important part in the angiogenesis and endometrial repair that occur after menstruation. This finding may have profound implications for understanding the etiology of endometriosis, as increased VEGF secretion in hypoxic tissue fragments could assist the vascularization of ectopic explants. VEGF expression is therefore controlled by multiple factors, including ovarian steroids, hypoxia, and other local regulators. It is likely that these act together during the menstrual cycle to produce the correct architecture of the endometrial vasculature.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received January 19, 1999.
Revised June 22, 1999.
Revised September 3, 1999.
Accepted September 14, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  X. Fan, S. Krieg, C. J. Kuo, S. J. Wiegand, M. Rabinovitch, M. L. Druzin, R. M. Brenner, L. C. Giudice, and N. R. Nayak VEGF blockade inhibits angiogenesis and reepithelialization of endometrium FASEB J, October 1, 2008; 22(10): 3571 - 3580. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Plaisier, E. Streefland, P. Koolwijk, V.W.M. van Hinsbergh, F.M. Helmerhorst, and J.J.H.M. Erwich Angiogenic Growth Factors and Their Receptors in First-Trimester Human Decidua of Pregnancies Further Complicated By Preeclampsia or Fetal Growth Restriction Reproductive Sciences, September 1, 2008; 15(7): 720 - 726. [Abstract] [PDF]
|
|
|
|
 |
|
 M. L. Hull, C. R. Escareno, J. M. Godsland, J. R. Doig, C. M. Johnson, S. C. Phillips, S. K. Smith, S. Tavare, C. G. Print, and D. S. Charnock-Jones Endometrial-Peritoneal Interactions during Endometriotic Lesion Establishment Am. J. Pathol., September 1, 2008; 173(3): 700 - 715. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Gashaw, S. Stiller, C. Boing, R. Kimmig, and E. Winterhager Premenstrual Regulation of the Pro-Angiogenic Factor CYR61 in Human Endometrium Endocrinology, May 1, 2008; 149(5): 2261 - 2269. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P.G. Groothuis, H.H.N.M. Dassen, A. Romano, and C. Punyadeera Estrogen and the endometrium: lessons learned from gene expression profiling in rodents and human Hum. Reprod. Update, July 1, 2007; 13(4): 405 - 417. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. W. Laschke and M. D. Menger In vitro and in vivo approaches to study angiogenesis in the pathophysiology and therapy of endometriosis Hum. Reprod. Update, July 1, 2007; 13(4): 331 - 342. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V Bourlev, N Volkov, S Pavlovitch, N Lets, A Larsson, and M Olovsson The relationship between microvessel density, proliferative activity and expression of vascular endothelial growth factor-A and its receptors in eutopic endometrium and endometriotic lesions. Reproduction, September 1, 2006; 132(3): 501 - 509. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Hickey, G. Krikun, P. Kodaman, F. Schatz, C. Carati, and C. J. Lockwood Long-Term Progestin-Only Contraceptives Result in Reduced Endometrial Blood Flow and Oxidative Stress J. Clin. Endocrinol. Metab., September 1, 2006; 91(9): 3633 - 3638. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Malik, K. Day, I. Perrault, D.S. Charnock-Jones, and S. K. Smith Reduced levels of VEGF-A and MMP-2 and MMP-9 activity and increased TNF-{alpha} in menstrual endometrium and effluent in women with menorrhagia Hum. Reprod., August 1, 2006; 21(8): 2158 - 2166. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Punyadeera, V.L. Thijssen, S. Tchaikovski, R. Kamps, B. Delvoux, G.A.J. Dunselman, A.F.P.M. de Goeij, A.W. Griffioen, and P.G. Groothuis Expression and regulation of vascular endothelial growth factor ligands and receptors during menstruation and post-menstrual repair of human endometrium Mol. Hum. Reprod., June 1, 2006; 12(6): 367 - 375. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. H. Y. Ng, C. C. W. Chan, O. S. Tang, W. S. B. Yeung, and P. C. Ho The role of endometrial and subendometrial vascularity measured by three-dimensional power Doppler ultrasound in the prediction of pregnancy during frozen-thawed embryo transfer cycles Hum. Reprod., June 1, 2006; 21(6): 1612 - 1617. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. J. Davie, E. V. Gerasimovskaya, S. E. Hofmeister, A. P. Richman, P. L. Jones, J. T. Reeves, and K. R. Stenmark Pulmonary Artery Adventitial Fibroblasts Cooperate with Vasa Vasorum Endothelial Cells to Regulate Vasa Vasorum Neovascularization: A Process Mediated by Hypoxia and Endothelin-1 Am. J. Pathol., June 1, 2006; 168(6): 1793 - 1807. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Gashaw, J. M. Hastings, K. S. Jackson, E. Winterhager, and A. T. Fazleabas Induced Endometriosis in the Baboon (Papio anubis) Increases the Expression of the Proangiogenic Factor CYR61 (CCN1) in Eutopic and Ectopic Endometria Biol Reprod, June 1, 2006; 74(6): 1060 - 1066. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. N. Jabbour, R. W. Kelly, H. M. Fraser, and H. O. D. Critchley Endocrine Regulation of Menstruation Endocr. Rev., February 1, 2006; 27(1): 17 - 46. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. O. D. Critchley, J. Osei, T. A. Henderson, L. Boswell, K. J. Sales, H. N. Jabbour, and N. Hirani Hypoxia-Inducible Factor-1{alpha} Expression in Human Endometrium and Its Regulation by Prostaglandin E-Series Prostanoid Receptor 2 (EP2) Endocrinology, February 1, 2006; 147(2): 744 - 753. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. H. Y. Ng, C. C. W. Chan, O. S. Tang, W. S. B. Yeung, and P. C. Ho The role of endometrial and subendometrial blood flows measured by three-dimensional power Doppler ultrasound in the prediction of pregnancy during IVF treatment Hum. Reprod., January 1, 2006; 21(1): 164 - 170. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Tesone, R. L. Stouffer, S. M. Borman, J. D. Hennebold, and T. A. Molskness Vascular Endothelial Growth Factor (VEGF) Production by the Monkey Corpus Luteum During the Menstrual Cycle: Isoform-Selective Messenger RNA Expression In Vivo and Hypoxia-Regulated Protein Secretion In Vitro Biol Reprod, November 1, 2005; 73(5): 927 - 934. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Koshiba, J. Kitawaki, M. Teramoto, Y. Kitaoka, H. Ishihara, H. Obayashi, M. Ohta, H. Hara, T. Adachi, and H. Honjo Expression of Allograft Inflammatory Factor-1 in Human Eutopic Endometrium and Endometriosis: Possible Association with Progression of Endometriosis J. Clin. Endocrinol. Metab., January 1, 2005; 90(1): 529 - 537. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N.J. Raine-Fenning, B.K. Campbell, N.R. Kendall, J.S. Clewes, and I.R. Johnson Endometrial and subendometrial perfusion are impaired in women with unexplained subfertility Hum. Reprod., November 1, 2004; 19(11): 2605 - 2614. [Abstract] [Full Text] |
), VEGF121
(
), VEGF165 (
), and VEGF189 (
).
