G protein-coupled estrogen receptor 1 (GPER, GPR 30) in normal human endometrium and early pregnancy decidua.

The recently identified trans-membrane G protein-coupled estrogen receptor 1 (GPER, GPR30) has been implicated in rapid non-genomic effects of estrogens. This focuses on expression and localization of GPER mRNA and protein in normal cyclic endometrium and early pregnancy decidua. Real-time PCR, western blotting, in situ hybridization and immuno-histochemistry were used. Endometrial expression of GPER mRNA was lower in the secretory phase than in the proliferative phase, and even lower in the decidua. The expression pattern was similar to that of ERα mRNA, but different from that of ERß mRNA. Western blot detected GPER protein as a 54 kDa band in all endometrial and decidual samples. In contrast to the mRNA, GPER protein did not show cyclic variations. Apparently, a lower amount of mRNA is sufficient to maintain protein levels in the secretory phase. GPER mRNA was predominantly localized in the epithelium of mid- and late-proliferative phase endometrium, whereas expression in early proliferative and secretory glands could not be distinguished from the diffuse stromal signal, which was present throughout the cycle. Immuno-staining for GPER was stronger in glandular and luminal epithelium than in the stroma throughout the cycle. The cyclic variations of GPER mRNA obviously relate to strong epithelial expression in the proliferative phase, and the expression pattern suggests regulation by ovarian steroids. GPER protein is present in endometrial tissue throughout the cycle, and the epithelial localization suggests potential functions during sperm migration at mid-cycle, as well as decidualization and blastocyst implantation in the mid-secretory phase.

Transvaginal ultrasound and lactate dehydrogenase isoenzyme activity profile in uterine aspirate for diagnosis of endometrial carcinoma in women with postmenopausal bleeding.

Postmenopausal patients with vaginal bleeding (n = 72) were evaluated with the combination of transvaginal ultrasonography (TVS) and analysis of the lactate dehydrogenase (LD) isoenzyme activity profile in uterine fluid aspirates. TVS evaluation of the endometrium was classified as <5 mm, > or =5 mm, or poorly defined. The LD isoenzyme activity profile was characterized as abnormal or normal. Pathologic findings were further evaluated with diagnostic curettage. TVS found the endometrium to be > or =5 mm or poorly defined in 44 patients (61%). Endometrial carcinoma was found in 6 of 72 patients (8%). They appeared in the TVS groups endometrium >5 mm (n = 2) and endometrium poorly defined (n = 4) but not in the endometrium <5 mm. The LD isoenzyme activity profile was abnormal in the six malignant cases and in ten benign cases. Thus, the need for further evaluation with hysteroscopy and curettage was reduced to 16 cases. Since TVS had 100% sensitivity but only 42% specificity, it is suitable for first-level examination in patients with postmenopausal bleeding. The second-level method should have similarly high sensitivity but much higher specificity. The LD isoenzyme activity profile in uterine fluid aspirates had 100% sensitivity and 85% specificity. Another important feature is that the method is not sensitive to endometrial thickness, amount of sample, sampling device, or dilution. Thus, it is more reliable than aspiration histology. For every hysteroscopy or curettage that can be replaced by LD analysis, the cost is reduced by approximately EUR 720 or 540, respectively.

Endometrial expression of the estrogen-sensitive genes MMP-26 and TIMP-4 is altered by a substitution protocol without down-regulation in IVF patients.

BACKGROUND: The aim of this study was to analyse the effects of an estradiol (E(2))-progesterone substitution protocol on the endometrial expression of estrogen-sensitive genes during the peri-implantation period. METHODS: Peripheral blood and endometrial biopsies were obtained from 13 infertile women both in a natural cycle (NC), on days 5 and 7 after ovulation (NC5, NC7), and in an artificial (substituted) cycle (AC), on days 5 and 7 of progesterone addition (AC5, AC7). Estrogen receptor-alpha (ERalpha) and progesterone receptor (PR) were assayed by immunohistochemistry. Matrix metalloproteinase-26 (MMP-26) mRNA and tissue inhibitor of metalloproteinase-4 (TIMP-4) mRNA were semiquantitatively assessed in tissue sections using in situ hybridization (ISH) and quantified in tissue extracts using real-time PCR. RESULTS: Levels of both E(2) and progesterone were higher in the peripheral blood in AC than in NC. Also on day AC5, expressions of ERalpha, PR and MMP-26 mRNA (focally) were increased in the epithelium and TIMP-4 mRNA in the stroma. Expression levels of these genes dropped significantly between AC5 and AC7, but not between NC5 and NC7. Abnormally high levels in AC5 samples suggest overstimulation with E(2), and the rapid decrease between AC5 and AC7 suggests overstimulation with progesterone. CONCLUSIONS: In ACs, increased levels of E(2) in the blood exaggerate the endometrial expression of estrogen-sensitive genes, whereas higher levels of progesterone in the blood in the secretory phase exaggerate the drop in expression of these genes. Dramatic variations in the gene expression may not be optimal for the implantation process.

Endometrial TIMP-4 mRNA is expressed in the stroma, while TIMP-4 protein accumulates in the epithelium and is released to the uterine fluid.

We have previously reported that endometrial mRNA expression of both tissue inhibitors of metalloproteinase-4 (TIMP-4) and matrix metalloproteinase-26 (MMP-26) peaks in the early secretory phase, which implies a role in implantation. The objective of this study was to compare the distribution of TIMP-4 and MMP-26 in endometrial tissue and uterine fluid over the menstrual cycle. Endometrial tissue was analysed with in situ hybridization and immunohistochemistry to localize mRNA and protein for TIMP-4 and MMP-26 in the same set of samples. TIMP-4 mRNA was quantified in separated stromal and epithelial cells using real-time PCR. Uterine fluid was analysed with western blotting. TIMP-4 mRNA was exclusively localized to the stroma, whereas MMP-26 mRNA was expressed by epithelial cells. TIMP-4 protein was only occasionally found in the stroma but was consistently present in granules of the apical part of luminal and glandular epithelial cells. TIMP-4, but not MMP-26, was demonstrated in uterine fluid. Thus, TIMP-4 is produced in the stroma only, secreted by stromal cells, taken up by epithelial cells, accumulated in apical granules and finally secreted to the uterine fluid. Maximal expression of MMP-26, and its strongest inhibitor TIMP-4, in the early and mid-secretory phase suggests a role during implantation. MMP-26 is stored in epithelial cells in its active form, is not released spontaneously and is controlled by TIMP-4 in both stroma and uterine fluid.

Implantation of the human embryo: research lines and models. From the implantation research network 'Fruitful'.

Infertility is an increasing problem all over the world, and it has been estimated that 10-15% of couples in fertile age have fertility problems. Likewise induced unsafe abortion is a serious threat to women's health. Despite advances made in assisted reproduction techniques, little progress has been made in increasing the success rate during fertility treatment. This document describes a wide range of projects carried out to increase the understanding in the field of embryo implantation research. The 'Fruitful' research network was created to encourage collaborations within the consortium and to describe our different research potentials to granting agencies or private sponsors.

[MMP-26 mRNA and estrogen receptor alpha co-expression in normal and pathological endometrium]. / Ko-exprese MMP-26 mRNaa estrogenového receptoru alfa v normálním a patologickém endometriu.

OBJECTIVE: To examine the expression pattern of matrix metalloproteinase-26 (MMP-26) mRNA and estrogen receptor-alpha (ER alpha) in normal, hyperplastic, premalignant and malignant endometrial tissue. DESIGN: Experimental study. SETTING: Department of Obstetrics and Gynecology of the Palacky University Medical School and University Hospital, Olomouc, Czech Republic, Department of Obstetrics and Gynecology, University Hospital, Lund, Sweden, Atherosclerosis Research Unit, King Gustav V Research Institute, Karolinska Hospital, Stockholm, Sweden. METHODS: We studied MMP-26 mRNA and ER alpha in 36 normal, 7 hyperplastic, 6 premalignant and 19 malignant endometrial samples. Based on histological examination, all normal specimens were classified according to an ideal 28 day menstrual cycle as early, mid, and late proliferative phase, early, mid and late secretory phase and menstrual phase. Samples with hyperplasia were classified as simple or complex. Premalignant samples were represented by complex hyperplasia with atypia. Malignant samples were histologically classified as well, intermediately and poorly differentiated, respectively. Specimens were analyzed using in situ hybridization and real time PCR. ER alpha was localized by immunohistochemistry. RESULTS: Epithelial MMP-26 mRNA expression was highest in the early secretory phase and in endometrial hyperplasia. Expression levels were low in the late secretory and menstrual phase and in malignant samples decreased gradually with dedifferentiation. Expression pattern of MMP-26 mRNA in normal, hyperplastic, premalignant and malignant endometrial tissue strongly co-variated with that of ER alpha. CONCLUSION: Co-expression of MMP-26 and ER alpha in normal and pathological endometrial tissue suggests possible regulation of MMP-26 gene by estrogen.

[Novel matrix metalloproteinases in cycling endometrium]. / Nové matrix metaloproteinázy v cyklickém endometriu.

OBJECTIVE: To examine the expression pattern of some novel matrix metalloproteinases (MMPs) in cycling endometrium. DESIGN: Experimental study. SETTING: Department of Obstetrics and Gynecology of the Palacky University Medical School and University Hospital, Olomouc, Czech Republic, Department of Obstetrics and Gynecology, University Hospital, Lund, Sweden. METHODS: We studied MMP-12, -16, -17, -19 and -26 mRNA in 39 normal endometrial samples obtained across the menstrual cycle. Based on histological examination, all specimens were classified according to an ideal 28 day menstrual cycle as early (n=8), mid (n=6) and late (n=7) proliferative phase, early (n=4), mid (n=4) and late (n=8) secretory phase and menstrual (n=3) phase. Cycle variation was examined in frozen samples using in situ hybridization. RESULTS: Three distinct pattern of MMP mRNA expression were detected in cycling endometrium. MMP-12 was expressed predominantly in perimenstrual period, MMP-16, -17 and 19 were expressed throughout the cycle and MMP-26 was found to be maximal in periovulatory period. CONCLUSION: Different endometrial expression patterns of novel MMPs during menstrual cycle may indicate their specific roles for menstruation, endometrial growth and remodelling and implantation.

Matrix metalloproteinase-26 (matrilysin-2) expression is high in endometrial hyperplasia and decreases with loss of histological differentiation in endometrial cancer.

OBJECTIVE: Matrix metalloproteinases (MMPs) are key players in the degradation of extracellular matrix and basement membranes, and are thus important in tumor invasion. Recently, MMP-26 (endometase), a novel matrilysin-type member of the MMP family, was cloned from an endometrial tumor. This study examines the expression of MMP-26 mRNA in hyperplastic, premalignant and malignant endometrial samples, and compares with normal endometrial tissue. METHODS: Endometrial carcinoma samples (19) were histologically classified as well, intermediately and poorly differentiated. Samples with hyperplasia (n = 15) were classified as simple, complex, or complex with atypia. Normal endometrial specimens (n = 39) were classified according to an ideal 28-day menstrual cycle and subsequently grouped in the early, middle, and late parts of the cycle. All samples were analyzed using in situ hybridization and real time PCR. The probes used for in situ hybridization and real time PCR recognized non-overlapping sequences. MMP-26 protein was localized by immunohistochemistry. RESULTS: MMP-26 mRNA was exclusively localized in the epithelial component of normal, hyperplastic, premalignant, as well as malignant samples. It was not found in the stroma of any tissue category. Quantifications with real time PCR as well as semi-quantifications of the in situ hybridization signal revealed maximal levels in normal tissue at midcycle and in endometrial hyperplasia both with and without atypia. The amount of MMP-26 mRNA decreased progressively with loss of histological differentiation in malignant samples. Immunostaining localized MMP-26 in epithelial glandular and luminal cells, in vessel walls, and in tumor cells. Since the pattern of MMP-26 expression mimicked that of ER-alpha, we searched the MMP-26 promoter region for a potential estrogen response element (ERE). A sequence at position -130 to -116 had high homology to the consensus sequence of an ERE. Based on these observations, we suggest that ER-alpha is involved in regulation of the MMP-26 gene. CONCLUSIONS: MMP-26 mRNA is selectively localized in the epithelial compartment of normal, hyperplastic, and malignant endometrial tissue. Expression is high in normal and hyperplastic endometria, but is downregulated in the late part of the cycle and in malignant tumors. The expression pattern of MMP-26 mRNA mimics that of ER-alpha, and the promoter region of the MMP-26 gene has a potential ERE.

Endometrial TIMP-4 mRNA is high at midcycle and in hyperplasia, but down-regulated in malignant tumours. Coordinated expression with MMP-26.

We have previously reported that endometrial expression of matrix metalloproteinase (MMP)-26 mRNA comes to a maximum in the early secretory phase. Since tissue inhibitor of metalloproteinase (TIMP)-4 is a potent inhibitor of MMP-26, the objective of this study was to identify the pattern of TIMP-4 mRNA expression in the normal endometrial cycle. We also evaluated hyperplastic, pre-malignant (atypical hyperplasia) and malignant endometrial tissue. Endometrial TIMP-4 mRNA was localized in tissue sections using in situ hybridization, and quantified in tissue extracts using real-time PCR. Estrogen receptor alpha (ERalpha) was assayed in the same set of samples using immunohistochemistry. In situ hybridization demonstrated TIMP-4 mRNA in the stroma of both normal and pathological tissues. TIMP-4 mRNA increased in the proliferative phase to a maximum in the early secretory phase, and then decreased in the late part of the cycle. Expression was comparable in normal and hyperplastic (including atypical) endometrial samples, whereas lower levels were detected in malignant tumours. Since this general pattern of expression suggests estrogen dependence, we evaluated ERalpha in our samples. Tissue sections from the normal proliferative phase, hyperplasia and pre-malignant atypical hyperplasia tissue stained strongly for ERalpha, whereas weak staining was seen in the secretory phase and in malignant tumours. Thus, low level of ERalpha was accompanied by down-regulated TIMP-4 mRNA, supporting the hypothesis that ERalpha contributes to regulation of the TIMP-4 gene. In addition, we identified a putative estrogen response element (ERE) in the promoter region of the TIMP-4 gene at position -930 to -916. Similarities in the cyclic patterns of TIMP-4 mRNA and MMP-26 mRNA, together with the fact that TIMP-4 is a potent inhibitor of MMP-26, suggest a functional relationship, and furthermore a role in human implantation.

Differential localization and expression of urokinase plasminogen activator (uPA), its receptor (uPAR), and its inhibitor (PAI-1) mRNA and protein in endometrial tissue during the menstrual cycle.

Normal endometrium is a highly dynamic tissue, which responds to ovarian steroids with cyclic proliferation, differentiation (secretion), and degradation (menstruation). The urokinase plasminogen activator (uPA)-dependent proteolytic cascade as well as ligand activation of the uPA receptor (uPAR) is critically involved in physiological as well as pathophysiological aspects of tissue expansion and remodelling. Cyclic variation and distribution of uPA, uPAR and plasminogen activator inhibitor 1 (PAI-1) mRNA were examined by in situ hybridization, real-time PCR and northern blot in normal endometrium. Their corresponding proteins were localized with immunohistochemistry. uPA mRNA is exclusively expressed by stromal cells, whereas uPA protein is present in both epithelial and stromal cells. Immunostaining for uPA protein is reduced or undetectable at midcycle, thus coinciding with peak concentration of uPA in the uterine fluid. uPAR mRNA is expressed by epithelial cells in the proliferative phase and by stromal cells in the secretory phase. However, epithelial cells stain for uPAR protein throughout the cycle, suggesting that uPAR may detach from stromal cells and then bind to epithelial cells in the secretory phase. PAI-1 mRNA is located in vessel walls. The late secretory phase has greatly increased expression of all three mRNA and their proteins, mainly in pre-decidual cells in the superficial stroma. Discordant localization of the mRNA and proteins suggest that uPA is produced by stromal cells, released and bound to epithelial cells in both the proliferative and secretory phases, whereas uPAR is released from the stroma and bound to epithelial cells in the secretory phase. Also, the present data together with earlier reports suggest that uPA is released from the epithelial cells to the uterine fluid.

Diagnosis of endometrial cancer in patients with postmenopausal bleeding by analysis of the lactate dehydrogenase isoenzyme activity profile in uterine fluid.

OBJECTIVES: We have previously shown that high activity of lactate dehydrogenase (LD) isoenzymes 4 and 5 in terine aspirates is a marker for endometrial carcinoma. The purpose of this study was to identify an abnormal activity profile of LD2-5 using LD1 as an internal standard, thereby being able to dispense with measurement of the total LD activity. The profile was subsequently tested for diagnostic power in clinical settings. METHODS: We used data from 11 cases of endometrial cancer. Each isoenzyme was estimated relative to the activity of LD1 (LD1 = 1.0). Based on the lowest level found for each of LD2-5 in the 11 cases, the cut-off levels for an "abnormal profile" were identified. The abnormal profile was subsequently tested for diagnostic power in a group of postmenopausal women at risk for endometrial cancer, that is, they had experienced vaginal bleeding (n = 100). A second group of asymptomatic postmenopausal women (n = 366) had endouterine aspiration performed as part of a regular gynecologic check up to evaluate the prevalence of an abnormal LD isoenzyme profile. RESULTS: In the group of postmenopausal women who had experienced vaginal bleeding, abnormal profile was found in 27 cases: 14 with adenocarcinoma and 13 with benign histology. All cases with normal profile had benign histology. Thus, sensitivity as well as the negative predictive value was 100%. In the group of asymptomatic women, abnormal LD isoenzyme profile was found in 15 cases (4.1%). All had benign histology. CONCLUSIONS: The LD isoenzyme profile detects endometrial malignancy with high accuracy, and equally important, a normal profile excludes malignancy. The profile, which uses relative rather than absolute activity levels, based on LD1 as an internal standard, has the great advantage of being independent of both the dilution factor and the aspiration technique. Larger studies comparing the LD isoenzyme activity profile with ultrasonographic evaluation and biopsy histology are needed.

[MMP-26 expression in endometrial explants treated with estradiol and progesterone]. / Exprese MMP-26 v endometriálních explantech pod vlivem estradiolu a progesteronu.

OBJECTIVE: To examine possible estrogen dependent endometrial expression of MMP-26 in vitro. DESIGN: Experimental study. SETTING: Department of Obstetrics and Gynecology of the Palacky University Medical School and University Hospital, Olomouc, Czech Republic, Department of Obstetrics and Gynecology, University Hospital, Lund, Sweden. METHODS: We studied MMP-26 mRNA in 14 normal endometrial samples obtained from the proliferative phase of the menstrual cycle. Samples were cultured for five days either with estradiol alone or in combination with progesterone. Samples cultured with ethanol represented control groups. MMP-26 mRNA expression was examined in frozen samples using in situ hybridization. Immunohistochemistry was used to study the presence of estrogen and progesterone receptors in endometrial explants. RESULTS: MMP-26 mRNA expression was highest in fresh (non cultured) samples. Signal intensity decreased during the first two days of culture and was negligable in the following days. Nuclear intensity for estrogen and progesterone receptor was high after five days of culture. CONCLUSION: We did not find MMP-26 mRNA in vitro expression to be directly estrogen dependent.

[Endometrial expression profiles of mRNA for selected matrix metalloproteinases and tissue inhibitor of metalloproteinases-1]. / Profily endometriální exprese mRNA u vybraných matrix metaloproteináz a tkánového inhibitoru metaloproteináz-1.

OBJECTIVE: To examine the endometrial expression pattern of messenger RNA (mRNA) for selected matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases-1 (TIMP-1). DESIGN: Experimental study. SETTING: Department of Obstetrics and gynecology of the Palacky University Medical School and University Hospital, Olomouc, Czech Republic, Department of Obsterics and Gynecology, University Hospital, Luna, Sweden. METHODS: We studied MMP-1, -3, -7, -10, -11, -12, -13, -14, -16, -26 and TIMP-1 mRNA in 39 normal endometrial samples obtained across the menstrual cycle. Based on histological examination, all specimens were classified according to an ideal 28 day menstrual cycle as early (n=8), mid (n=6) and late (n=7) proliferative phase, early (n=4), mid (n=4) and late (n=8) secretory phase and menstrual (n=3) phase. mRNA extracted from frozen tissue samples was quantitated using real time PCR. RESULTS: Four distinct patterns of MMP mRNA expression were detected in cycling endometrium. MMP-1, -3, -10, and -12 were expressed predominantly in perimenstrual period, MMP-7 and -11 had highest levels in proliferative phase, MMP-13, -14 and -16 were expressed throughout the cycle and MMP-26 was found to be maximal in periovulatory period. Levels of TIMP-1 mRNA remained unchanged during the cycle. CONCLUSION: The specific endometrial expression profiles of MMPs during menstrual cycle point to their specific biologic roles during the cycle. MMP-26 exhibits a unique expression pattern.

Epithelial expression of matrix metalloproteinase-26 is elevated at mid-cycle in the human endometrium.

The human endometrium is a dynamic tissue, which undergoes extensive tissue remodelling during the menstrual cycle. Due to their involvement in such processes, several well-characterized matrix metalloproteinases (MMP) have previously been studied in the endometrium. MMP-26 is a newly described matrilysin. We studied MMP-26 mRNA in 39 normal endometrial samples obtained across the menstrual cycle. Tissue distribution and cycle variation was examined using in-situ hybridization, Northern blot analyis and real time PCR. The probes for Northern blot analysis and real time PCR recognized non-overlapping sequences. MMP-26 was localized exclusively in epithelial cells of both glands and the luminal surface. Expression increased during the proliferative phase to a maximum at mid-cycle, then decreased to non-detectable levels in the late secretory and menstrual phases. Expression of MMP-26 mRNA in endometrial tissue explants in vitro required stimulation with both estradiol and progesterone. The tissue content of c-jun mRNA was assayed, since c-jun, as part of the enhancer complex AP-1, may be involved in regulation of MMP-26 gene transcription. The pattern of c-jun expression over the menstrual cycle was similar to that of MMP-26. Epithelial expression in the peri- and post-ovulatory stages of the menstrual cycle suggests the involvement of MMP-26 in reproductive processes.

Urokinase plasminogen activator and its inhibitor, PAI-1, in association with progression-free survival in early stage endometrial cancer.

Components of the urokinase plasminogen activator (u-PA) system are involved in the metastatic process, and have accordingly been associated with clinical outcome in a variety of malignant tumours. We investigated the prognostic importance of u-PA and plasminogen activator inhibitor type 1 (PAI-1) in endometrial cancer, analysed with luminometric immunoassay (LIA) and enzyme-linked immunosorbent assay (ELISA), respectively. Two different cut-off levels were used: the median and the 80th percentile-the latter because of the low progression rate for patients with early stage (I-II) endometrial cancer. After a median follow-up time of 6.8 years, univariate analysis of patients with stage I-II disease (n=188) showed that high u-PA and high PAI-1 content was associated with a shorter progression-free survival (PFS), but at different cut-off levels, uPA at the median (P=0.003), and PAI-1 at the 80th percentile (P<0.001). Among the other factors, DNA ploidy status was most strongly correlated to PFS, followed by age (continuous), International Federation of Gynaecology and Obstetrics (FIGO) grade of differentiation, S-phase fraction and progesterone receptor (PgR) status. Bivariate analyses, including ploidy and one of the factors u-PA or PAI-1, showed that both add significant prognostic information. We conclude that u-PA and PAI-1 are promising prognostic factors in early stage endometrial cancer.

Dedifferentiation of serous ovarian cancer from cystic to solid tumors is associated with increased expression of mRNA for urokinase plasminogen activator (uPA), its receptor (uPAR) and its inhibitor (PAI-1).

The plasminogen activating system is involved in tumor growth and metastasis by degradation of extracellular matrix, and modulation of cell adhesion and migration. Benign and well-differentiated malignant ovarian tumors present as cystic lesions with preserved glandular morphology, whereas poorly differentiated tumors and metastases are solid with characteristic absence of glandular morphology. We analyzed the mRNAs for urokinase plasminogen activator (uPA), its receptor (uPAR), and inhibitor (PAI-1) in serous ovarian tumors by in situ hybridization and by densitometric scanning of Northern blots prepared from tissue extracts. The mRNA expressing cells in the in situ hybridization sections were evaluated and counted by two different observers. The number of mRNA expressing cells for uPA, uPAR and PAI-1 were all significantly increased in solid as compared with cystic malignant tumors. The increased expression of all three mRNA species was mainly located in the stroma of poorly differentiated tumors and metastases. Apart from being expressed in the stroma of these tumors, uPAR mRNA was also expressed by tumor cells located along the stromal/epithelial boarder. In addition, the tumor tissue content of uPA, uPAR and PAI-1 mRNAs as measured by Northern blots were higher in the solid as compared with the cystic tumors. Increased expression of uPA, uPAR and PAI-1 genes in the solid tumors suggest a correlation with a more aggressive phenotype.

Paracrine stimulation of capillary endothelial cell migration by endometrial tissue involves epidermal growth factor and is mediated via up-regulation of the urokinase plasminogen activator receptor.

Endometrial angiogenesis is not well studied, but has potential as a model for studies of physiological angiogenesis. Migration as well as proliferation of vascular endothelial cells are modulated by other endometrial cells. This study analyzes the chemotactic signal released from endometrial tissue in a wound assay using human microvascular endothelial cells. Endometrial tissue explants stimulate migration, and this effect is significantly weaker with explants taken at midcycle than those obtained earlier or later in the cycle. Migration is inhibited more than 50% by either blocking antibodies to the urokinase plasminogen activator receptor (uPAR) or enzymatic removal of uPAR from the cell surface. Also, migration is inhibited more than 50% by antibodies to epidermal growth factor (EGF), but not by antibodies to vascular endothelial growth factor or basic fibroblast growth factor. The combination of anti-EGF and anti-uPAR antibodies does not further reduce the response, suggesting that these antibodies target a common pathway. Conditioned medium from endometrial explants contains EGF, and EGF stimulates the migration of endothelial cells in a dose-dependent way. This effect is completely blocked by antibodies to uPAR. These data suggest up-regulation of the uPA system by EGF. Conditioned medium from EGF-treated cells contains less uPA than medium from control cells. In contrast, binding of radiolabeled uPA reveals an increased number of uPA-binding sites in EGF-treated cells. Increased expression of uPAR potentially increases the activation and assembly of focal adhesion sites, a prerequisite for cell migration. We conclude that the endometrial migratory signal has two components. The major part of the signal is blocked by antibodies to EGF, and the response is mediated via up-regulation of uPAR in the endothelial cells. The other part of the signal is unknown, and the response does not involve uPAR. Decreased endometrial chemotactic signal at midcycle is probably related to decreased release of EGF, which is secondary to increased binding to endometrial cell membranes.

High tissue content of urokinase plasminogen activator (u-PA) is associated with high stromal expression of u-PA mRNA in poorly differentiated serous ovarian carcinoma.

Urokinase plasminogen activator (u-PA) plays a pivotal role in tissue degradation during tumor spread and metastasis. We have quantitated u-PA in tissue homogenates of 31 serous ovarian tumors and localized u-PA and its mRNA in tissue sections of 26 serous ovarian tumors. The content of u-PA was higher in malignant than in benign tumors, with the highest levels being found in poorly differentiated cancers. In tissue sections, the u-PA mRNA was hybridized with a radiolabeled RNA probe. Signals were almost exclusively found in the epithelium in benign and borderline tumors and in well-differentiated cancers. Poorly differentiated tumors and metastases exhibited prominent stromal expression of u-PA mRNA, whereas epithelial expression was weak or absent. Immuno-histochemical staining co-localized u-PA antigen with its mRNA in the epithelium of benign and borderline tumors and in well-differentiated cancers. Poorly differentiated malignant tumors showed extensive immunostaining in the epithelium in addition to stromal staining. The u-PA mRNA-expressing and u-PA-immunostained cells in the stroma were not tumor cells since no cells in the stroma were positive for cytokeratin. Poorly differentiated tumors had increased numbers of stromal macrophages (CD68), and they co-localized with some of the u-PA-positive cells. The presence of u-PA antigen and the absence of u-PA mRNA in tumor epithelium of poorly differentiated tumors and metastases together with the presence of u-PA mRNA in the stroma suggests production in stromal cells and subsequent binding to receptor sites in tumor cells.

Human endothelial cell migration is stimulated by urokinase plasminogen activator:plasminogen activator inhibitor 1 complex released from endometrial stromal cells stimulated with transforming growth factor beta1; possible mechanism for paracrine stimulation of endometrial angiogenesis.

Human endometrial stromal cell cultures, stimulated for two days with recombinant transforming growth factor beta1 (TGFbeta1; 10 ng/ml), contained conditioned medium concentrations of urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI1), and uPA:PAI1 complex. Since a number of cellular effects have been reported to follow a binding of enzymatically inactive uPA to the receptor in different cell types, we studied the influence of uPA:PAI1 complex on human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC-1). Increasing concentrations of uPA:PAI1 complex as well as free uPA resulted in a dose-dependent stimulation of endothelial cell migration. Stimulation by the complex was of the same magnitude as that of free uPA on a molar basis and reached its maximum at 1 nM in both cell types. PAI1 by itself, however, had no effect on cell migration. The migratory response to both uPA and the uPA:PAI1 complex was inhibited by antibody adhesion to the cell surface receptor for uPA. In addition, we found that TGFss1 had a direct stimulatory effect on migration in both HUVEC and HMEC-1. This response did not, however, involve the binding of uPA to the uPA receptor. Since TGFbetas are expressed in endometrial tissue and reportedly stimulate angiogenesis in other tissues in vivo, though not endothelial cell proliferation in vitro, they may engage in the regeneration of endometrial vasculature indirectly via perivascular cells. We found that the uPA:PAI1 complex, when released from endometrial stromal cells in response to TGFbeta1, stimulated endothelial cell migration. This suggests a possible mechanism for paracrine stimulation of endometrial angiogenesis.

Soluble receptor for urokinase plasminogen activator in both full-length and a cleaved form is present in high concentration in cystic fluid from ovarian cancer.

We assayed the levels of soluble urokinase plasminogen activator receptor (su-PAR) with an ELISA in various body fluids from 77 patients with benign or malignant ovarian tumors, histologically classified as follows: benign; possibly malignant (borderline); and well, intermediately, and poorly differentiated malignant. The concentration of su-PAR in fluid from malignant cysts was extremely high, approximately 10-fold higher than the concentration in ascitic fluid and approximately 100-fold higher than that in blood. Also, the concentration in malignant cysts was approximately 10-fold higher than the concentration in benign cysts. Such high concentrations of su-PAR were found not only in truly malignant but also in possibly malignant cysts. Thus, we suggest that the concentration of su-PAR in cystic fluid can serve as a marker, allowing early diagnosis of malignant ovarian cysts. The concentration of su-PAR in fluid aspirated transvaginally with ultrasonographic guidance can be used to discriminate possibly and truly malignant cysts from benign cysts without surgery. The high concentrations of su-PAR in ovarian cystic fluids allowed us to characterize the molecular forms. Cross-linking of a radiolabeled ligand to the receptor demonstrated that at least a fraction of su-PAR was able to bind the ligand. A cleaved form of the receptor constituting domains 2 and 3 was detected with Western blotting. The cleaved receptor, which is devoid of the ligand-binding domain 1, has not previously been demonstrated in body fluids; it has only been demonstrated on cell surfaces. su-PAR domains 2 and 3 were found in cystic fluids from both malignant and benign ovarian tumors.