Mol. Hum. Reprod. Advance Access originally published online on January 29, 2004
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Molecular Human Reproduction, Vol. 10, No. 3, pp. 159-166, 2004
© European Society of Human Reproduction and Embryology 2004
In situ localization of mRNA for the fibrinolytic factors uPA, PAI-1 and uPAR in endometriotic and endometrial tissue
1Department of Obstetrics and Gynaecology, Huddinge University Hospital, SE-141 86 Stockholm and 2Department of Clinical Neuroscience, Section of Psychiatry, Karolinska Institutet, SE-171 76 Stockholm, Sweden
3 To whom correspondence should be addressed. e-mail: christine.bruse{at}hs.se
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ABSTRACT |
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Endometriotic tissue grows invasively. The plasminogen-activating system is suggested to participate in degradation of extracellular matrix (ECM) and modulation of cell adhesion and migration. We have previously demonstrated elevated levels of the fibrinolytic factors urokinase plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1) in endometriotic tissue and endometrium from women with endometriosis. The aim of the present study was to localize the uPA, PAI-1 and urokinase plasminogen activator receptor (uPAR) mRNA in endometriotic tissue and in endometrium both from women with and without endometriosis. With in situ hybridization, we found that uPA mRNA seems to be up-regulated in endometriotic glands and endometrial stroma as well as PAI-1 mRNA in endometriotic and endometrial stroma from women with endometriosis. uPAR mRNA likewise appears to be up-regulated in both glands and stroma in endometriotic tissue and in endometrial glands from patients compared to endometrial glands and stroma from healthy women. These differences might be important for menstrual shedding and adherence of endometrial fragments to peritoneal lining in women developing endometriosis and for the invasive growth of endometriotic tissue.
Key words: Key words: endometriosis/endometrium/fibrinolytic factors/mRNA
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Introduction |
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Endometriosis is a common gynaecological disorder affecting women of fertile age with pelvic pain and infertility problems, and is characterized by the presence of endometrial glands and stroma outside the uterine cavity. After adherence of retrogradely shed endometrial fragments to the peritoneum, endometriotic tissue has the ability to grow invasively (Spuijbroek et al., 1992). Similar to neoplastic growth, the plasminogen-activating system may participate in this process.
The plasminogen-activating system, including both the plasminogen activators and their inhibitors, is involved in tissue degradation and remodelling under both normal and pathological conditions (Danø et al., 1985). Normal conditions include mammary gland involution, ovulation, blastocyst implantation and wound healing. Pathological conditions include tumour growth, invasion, and metastasis (Andreasen et al., 2000). The plasminogen-activating system also appears to be involved in other cancer cell-directed tissue-remodelling processes, such as angiogenesis and stimulation of fibroblast proliferation and extracellular matrix (ECM) protein synthesis (Andreasen et al., 2000). The activation of plasminogen, leading to the formation of plasmin, is catalysed by urokinase plasminogen activator (uPA) when bound to uPA receptor (uPAR), or by tissue-type PA (tPA). uPA binds to uPAR with high affinity. Receptor binding of uPA initiates pericellular proteolysis and cell migration, two processes required for tissue invasion (Danø et al., 1994; Andreasen et al., 2000). Two plasminogen activator inhibitors (PAI), PAI-1 and PAI-2, regulate the plasminogen-activating system.
Plasmin, a highly potent protease, is able to degrade a broad spectrum of matrix and basement membrane proteins, such as fibronectin, laminin and proteoglycan (Mignatti et al., 1993). Plasmin also activates zymogens of other matrix-degrading proteases such as collagenases, stromolysins and elastases, i.e. matrix metalloproteinases (MMP) (Matrisian, 1992). Further, it catalyses activation of latent transforming growth factor-ß, (TGF-ß), important for the up-regulation of PAI-1 (Mignatti et al., 1993; Casslén et al., 1998).
In a previous study (Bruse et al., 1998), we assayed the PA and PAI in homogenates of endometriotic tissue and endometrium from women with endometriosis and from healthy controls. We found significantly higher levels of uPA and PAI-1 in endometriotic tissue and endometrium from women with endometriosis compared with controls. PAI-1 levels were even higher in the endometriotic lesions than in the endometrium from the same women.
We have recently found that separated stromal cells from endometriotic tissue and endometrium, cultured in macrophage-, hormone- and growth factor-free medium, released uPA, PAI-1 and soluble urokinase plasminogen activator receptor (suPAR). The release of PAI-1 was significantly higher from endometriotic stromal cells than from the other cell types. We also found that addition of 17ß-estradiol, progesterone or raloxifen to cultured endometrial and ovarian endometriotic stromal cells changed their release of uPA and PAI-1 but not of suPAR, compared to basal release. The uPA release decreased significantly in progesterone-treated endometrial stroma cells, while PAI-1 was significantly increased under the same conditions (Guan et al., 2002).
Sillem et al. (1997) reported that the suPAR release in endometrial cell cultures from women with endometriosis was significantly higher compared to controls. In these unseparated cell cultures, synthetic hormones such as promegestone or diethylstilbestrol did not influence the release.
In view of these findings, we decided to investigate by in situ hybridization (ISH) whether and in which type of cells, and in which menstrual phases, mRNA for uPA, PAI-1 and uPAR were expressed in paired endometriotic and endometrial samples, and to compare these mRNA levels with endometrial samples from healthy controls.
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Materials and methods |
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Sources of tissue for uPA mRNA localization
Twelve samples of endometriotic tissue (six from endometriotic cysts, three peritoneal biopsies, two ovarian biopsies, and one from a scar in the abdominal wall), were obtained from 12 women (mean age 32 years; range 25–47 years) at surgery for clinical reasons. An endometrial sample from each one of the women with endometriosis was obtained simultaneously by uterine curettage. Three additional endometrial samples from women with endometriosis (mean age 29 years; range 24–32 years) were obtained at surgery for clinical reasons. Endometrial samples obtained by uterine curettage from 14 healthy women (mean age 37 years; range 25–47 years) undergoing laparoscopic sterilization (n = 12), cone biopsy (n = 1) or surgery for a benign cyst (n = 1) were used as controls.
Six of the women with endometriosis were in the early and one in the late proliferative phase. A further three women were in the early and four in the late secretory phase and one was in the menstrual phase. Six women from the control group were in the early and one in the late proliferate phase, and three were in the early and four in the late secretory phase.
Sources of tissue for PAI-1 mRNA localization
Ten samples of endometriotic tissue (six from endometriotic cysts, three peritoneal biopsies, and one from a scar in the abdominal wall), were obtained from 10 women (mean age 33 years; range 25–47 years) at surgery for clinical reasons. From all of these women, an endometrial sample was obtained simultaneously by uterine curettage. From 10 healthy women (mean age 39 years; range 36–47 years) undergoing laparoscopic sterilization (n = 9) or surgery for a benign cyst (n = 1), 10 endometrial samples were obtained by uterine curettage and used as controls.
Four of the women with endometriosis were in the early and one in the late proliferative phase of the menstrual cycle, and three in the early and two in the late secretory phase of the menstrual cycle. Five women from the control group were in the early proliferative phase, none in the late proliferative phase, two in the early and three in the late secretory phase.
Sources of tissue for uPAR mRNA localization
Samples of endometriotic tissue (five from endometriotic cysts, three peritoneal biopsies, and one ovarian biopsy) were obtained from nine women (mean age 36 years; range 25–47 years) at surgery for clinical reasons. From eight of these women, an endometrial sample was obtained simultaneously by uterine curettage. Moreover, we obtained endometrial samples from another 10 women with endometriosis (mean age 32 years; range 25–43 years). Endometrium obtained by uterine curettage from 13 healthy women undergoing surgery for benign reasons [laparoscopic sterilization (n = 7), surgery for a benign cyst (n = 3), cone biopsy (n = 1), hysteroscopy (n = 1) or myoma surgery (n = 1)] served as controls.
Five of the 19 women in total with endometriosis were in the early and five in the late proliferative phase; three were in the early and four in the late secretory phase; two were in the menstrual phase. Five women from the control group were in the early and two in the late proliferative phase; two were in the early and four in the late secretory phase.
None of the women had taken any sex steroid hormones, been pregnant or breastfeeding the previous 2 months before surgery. The local ethics committee at Huddinge University Hospital had approved the study and the women had given their oral informed consent for the samples to be collected. Institutional review board approval is not required in Sweden.
Sample preparation
All tissue samples were rinsed in cold saline, immediately snap-frozen on dry ice or in liquid nitrogen, and stored at–70°C until sectioning within 2 years. The tissues were sectioned at 14 µm in a cryostat microtome (Microm HM 500M; Microm Laborgeräte GmbH, Germany) at –18°C, thaw-mounted on silane-coated glass slides, and stored at –70°C until hybridization was performed.
The phase of the menstrual cycle was determined according to the histological phase pattern of the endometrium (Noyes et al., 1950), and when available the plasma level of estradiol and progesterone at the time of surgery. Dating of endometriotic tissues was not performed.
cRNA probes
Human uPA cDNA, bp 1–1340 cloned into the PstI site of pBluescript SK-UK 8 (Verde et al., 1984), was used. The plasmid was provided by Prof. F.Blasi (Milan, Italy). Templates for generating antisense cRNA probes were prepared by linearizing uPA cDNA with XbaI (Pharmacia Biotech, Sweden) digestion. Sense control cRNA probes were prepared by analogous linearization with SalI (Pharmacia Biotech).
cDNA for human uPAR, bp 184–451 cloned into the PstI site of pBluescript KS+, (Pyke et al., 1991), was provided by Dr K.Danø (Copenhagen, Denmark). Templates for generating antisense cRNA probes were prepared by linearizing uPAR with SacI (Pharmacia Biotech) digestion, and sense control cRNA probes were prepared with KpnI (Pharmacia Biotech).
Human PAI-1 cDNA, bp 1–2876 cloned into the EcoRI site of pGEM-1 (Ny et al., 1986), was provided by Dr T.Ny (Umeå, Sweden). Templates for generating antisense cRNA probes were prepared by linearizing PAI-1 with HincII (Pharmacia Biotech) digestion. Sense control cRNA probes were prepared with DraIII (New England Biolabs, USA).
Linearization was confirmed by agarose gel (Sigma, USA) electrophoresis. As a molecular weight standard, a 1 kb DNA ladder (Invitrogen, USA) was used for each gel. The cRNA probes were labelled with 35S-UTP (Du Pont NEN, USA) specific activity 12.5 mCi/ml. The following RNA polymerases (Ambion Inc., USA) were used: antisense T7 and sense T3 for uPA, antisense T3 and sense T7 for uPAR, and antisense T7 and sense SP6 for PAI-1. The uPA probe was reduced by limited alkaline hydrolysis at pH 10.2.
In situ hybridization
All tissue samples for each mRNA species were run together under identical conditions. Prior to hybridization, tissue sections were pre-treated essentially as previously described by Young (1990). Briefly, the sections were fixed in 4% formaldehyde for 5 min. After rinsing in phosphate-buffered saline (PBS) for 3 min, the slides were deproteinized in 0.1 mol/l HCl for 20 min. After a short rinse in PBS, the sections were acetylated in 0.1 mol/l triethanolamine pH 8.0, and 0.25% acetic anhydride for 20 min. After rinsing twice in PBS, the sections were dehydrated stepwise in ethanols, 70, 80 and 100%, for 2 min, delipidated in chloroform for 5 min and rinsed in ethanol 100% twice for 2 min and then air-dried. The cRNA hybridization histochemistry was performed as described by Cox et al. (1984) and Whitfield et al. (1990). The final hybridization buffer, with 10x106 c.p.m. probe per ml, consisted of 2% 1 mol/l Tris–HCl (pH 8.0), 0.4% 0.5 mol/l ethylenediaminetetra-acetic acid disodium salt (EDTA) (pH 8.0), 6.7% 5 mol/l sodium chloride, 50% formamide, 20% 50xdextran sulphate, 2% 50xDenhardt’s solution, 4% 5 mol/l dithiothreitol (DDT), and 2% yeast tRNA (Pharmacia Biotech) (25 mg/ml). The labelled cRNA probes in hybridization buffer were denatured at 65°C for 5 min. Hybridization buffer (110 µl/12 cm2) was applied to tissue sections on each slide and covered with untreated glass cover slips. The slides were incubated at 55°C in chambers humidified with 2xsaline sodium citrate (SSC)/50% formamide overnight. After hybridization, the sections were washed four times in 2xSSC for 5 min, and immersed in RNase (Pharmacia Biotech) (10 mg/l) for 30 min at 37°C. The sections were de-salted in graded SSC solutions with 5 mol/l DDT, and washed once in 0.1xSSC at 65°C for 30 min and at room temperature for 5 min (Simmons et al., 1989). Thereafter the sections were dehydrated in a graded series of ethanols, 70, 80 and 99%, for 1 min, air-dried and exposed to Amersham HyperfilmTM-MP (Aylesbury, UK) for 5 days for signal detection. After that, the slides were coated with nuclear track emulsion NTB-2, (Eastman Kodak, USA) undiluted for uPA and PAI-1, and diluted 50% for uPAR. Following exposure for 3–14 weeks at 4°C, the slides were developed in D-19 Developer (Eastman Kodak Rochester) and counterstained with Cresyl Violet (Histolab, Sweden). The sections were finally analysed by using a Nikon UFX-DX microscope with light- and dark-field illumination. Hybridization with the sense probe for uPA, PAI-1 and uPAR showed only background signals.
We used a semiquantitative, four-grade scale for evaluation of the mRNA expression in the tissues as follows. – = no specific mRNA hybridization signal; + = weak mRNA hybridization signal; ++ = moderate mRNA hybridization signal; +++ = strong mRNA hybridization signal.
Statistics
For statistical calculations, no specific mRNA hybridization signal = 0, weak = 1, moderate = 2, and strong = 3. Differences in hybridization signals between the three types of tissues were analysed by Kruskal–Wallis test, followed by post hoc analysis with Mann–Whitneys U-test. P < 0.05 was considered statistically significant.
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Results |
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uPA mRNA
The results are given in Table I. In endometriotic tissue, stromal cells from all samples and glands from nine of 12 samples expressed hybridization signals for uPA mRNA. The signals varied from weak to moderate both in glands and stroma. Weak-to-strong signals for uPA mRNA were expressed in endometrial stroma from all women with or without endometriosis. However, in glands, weak mRNA signals were expressed only in one sample from each group of endometrium, both of them in early proliferative phase. The expression pattern varied between individual cells, both in glands and stroma. In endometriotic glands and stromal cells (Figure 1a) and in endometrial stromal cells from women with endometriosis (Figure 1b), the signals were frequently localized in clusters. In endometrial stromal cells from controls, the signal pattern was more dispersed (Figure 1c). There were no statistically significant differences between the cycle phases, either in glands or in stroma, in the three types of tissues. There were, however, statistically significant differences in the expression of signals when the three types of tissue were compared (Table II). There were statistically significant differences between endometriotic glands and endometrial glands both in endometrium from women with endometriosis and without, when both proliferative and secretory phases were compared as well as only comparison of secretory phases (P < 0.01, P < 0.001, P < 0.01 and P < 0.01 respectively). Stromal cells expressed significantly stronger signals for uPA mRNA in endometrium from women with endometriosis than stromal cells from endometrium from controls when comparing both proliferative and secretory phases (P < 0.05).
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PAI-1 mRNA
The results are given in Table III. Glands did not express any specific hybridization signal for PAI-1 mRNA in any of the three tissue types. Stromal cells expressed signals in all samples, except one each of endometriotic tissue and endometrium from a patient with endometriosis and four control endometrium. The signals varied from weak to strong expression. Both endometriotic and endometrial stroma from women with endometriosis expressed significantly stronger signals for PAI-1 mRNA than endometrial stroma from controls (P < 0.01and P < 0.05 respectively; Table IV). When comparing phases, endometrium from women with endometriosis expressed hybridization signals with significantly higher intensity in the proliferative phase (P < 0.05) than endometrium from controls. In secretory phase, endometriotic stroma expressed signals with significantly higher intensity than endometrial stroma from controls (P < 0.05). In endometriotic and in endometrial stroma from women with endometriosis (Figure 2a,b) the hybridization signals were frequently localized in clusters, while in endometrial stroma from controls (Figure 2c), the signals were more dispersed. There were no statistically significant differences in the intensity of hybridization signals between cycle phases in the three types of tissue.
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uPAR mRNA
The results are given in Table V. Both glands and stroma in all samples from endometriotic tissue expressed hybridization signals for uPAR mRNA from weak to strong intensity (Figure 3a). In endometrium from women with endometriosis, glands and stroma in all samples in the proliferative phase expressed hybridization signals, whereas signals were absent in glands and stroma in two and one sample respectively in secretory phase (Figure 3b). In four samples, two in proliferative and two in secretory phase, the glandular cells expressed signals with extraordinarily varying intensity, from weak to strong. In endometrium from controls, the signal for uPAR mRNA was absent in glands in four of six samples in secretory phase as well as in stroma in two of seven samples in proliferative phase. All other control samples were positive although the signals were weak (Figure 3c). In proliferative and secretory phase, endometriotic stromal cells expressed significantly stronger hybridization signals for uPAR mRNA than endometrial stromal cells from control women (P < 0.05, P < 0.05) and in proliferative phase significantly stronger than in endometrial stromal cells from women with endometriosis (P < 0.05) (Table VI). There were no statistically significant differences in the expression of signals between the cycle phases in stroma from the three types of tissue or in glands in endometriotic tissue. However, in endometrial glands from women with endometriosis and in controls, the expression for uPAR mRNA was significantly stronger in proliferative phase than in secretory phase for both (Table VI).
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Discussion |
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As far as we know from the literature, this is the first study on in situ localization for uPA, uPAR and PAI-1 mRNA not only in endometriotic tissue but also in endometrial tissue from women with endometriosis compared to endometrium from healthy women.
Endometriotic glands were uPA mRNA positive to a higher degree than endometrial glands, both from women with endometriosis and from controls. uPA mRNA expression was stronger in endometrial stroma from women with endometriosis throughout the menstrual cycle compared with endometrial stroma from controls. Also the mRNA pattern differed between the tissue types, being more dispersed in control endometrium. In stromal cells from women with endometriosis, the uPA mRNA was more localized in clusters. The cluster formation may represent one single cell, but the method did not allow such discrimination between cells in the formation. This difference might indicate an up-regulation of uPA mRNA in endometriotic and endometrial stromal cells from women with endometriosis, resulting in the higher uPA protein level previously observed (Bruse et al., 1998). This finding may be compared to studies on endometrial carcinomas, where the expression of uPA mRNA was significantly higher in all grades of tumour than in normal endometrium (Foca et al., 2000). uPA protein levels were, however, not measured in that study. We could not find any statistically significant difference between phase of the cycle in the three types of tissue tested. This is in accordance with the findings by Foca et al. (2000). However, Nordengren et al. (1998) found a discordance between uPA mRNA and uPA protein in endometrial malignancy, i.e. the mRNA level being unchanged, while the protein was significantly increased in malignant tissue compared to normal. Known inducers of the uPA gene are growth factors, peptide and steroid hormones, UV light, and cell morphology changes (Irigoyen et al., 1999).
PAI-1 mRNA was only expressed in stromal cells in the three tissue types. In accordance with our previous study of fibrinolytic factors in tissue homogenates, PAI-1 levels were higher in endometriotic and endometrial tissue from women with endometriosis compared to controls. Growth factors such as TGF-ß1, EGF and bFGF are known inducers of PAI-1 mRNA (Sandberg et al., 1997; Irigoyen et al., 1999), as are cytokines such as IL-1 and TNF-
and also over-expression of p53 tumour suppressor (Kunz et al., 1995). All these factors have been identified in the human endometrium (Rusnati et al., 1990; Chegini et al., 1992; Gold et al., 1994). Also progesterone has been presumed to be an inducer of PAI-1 mRNA in endometrial stromal cells (Casslén et al., 1992, 1995; Schatz et al., 1993), but later in vitro studies (Sandberg et al., 1997) showed that growth factors such as TGF-ß1 are probably the inducers of the gene and that progesterone increases the stability of PAI-I mRNA, resulting in increased levels of the PAI-1 protein in the secretory phase. Progesterone significantly increases the PAI-1 release in cultured endometrial but not in endometriotic stromal cells (Guan et al. 2002). TGF-ß1, on the other hand, had no effect on the release of PAI-1 from endometrial stromal cells whereas the release from endometriotic stromal cells was significantly increased (Guan et al. 2003). TGF-ß mRNA is present in the endometrial tissue throughout the menstrual cycle (Chegini et al., 1994; Gold et al., 1994), with increased levels from mid-secretory to menstrual phases (Casslén et al., 1998). IL-1 increases not only PAI-1 mRNA expression in human endometrial stromal cells, but also uPAR and suPAR expression in a dose-dependent manner (Chung et al., 2001). We have previously shown that the IL-1ß protein concentration is significantly higher in endometriotic tissue than in endometrium both from patients with endometriosis and from healthy controls (Bergqvist et al., 2001). IL-1ß levels were significantly higher in the secretory phase in endometrium from women with endometriosis.
Other cells releasing uPA and PAI-1 are inflammatory cells, mainly macrophages. In both tissue types from women with endometriosis, the mRNA for uPA and PAI-1 was more often expressed in clusters in endometriotic and endometrial stroma from patients compared to controls. This might indicate mRNA production from inflammatory cells within the stroma, which has to be studied further by immunohistochemistry.
The cluster formation, however, did not show any cycle dependence. Leukocyte infiltration in normal endometrial stroma begins at the end of the secretory phase and disappears in early proliferation (Noyes et al., 1950). Macrophages in human endometrium, both from women with and without endometriosis, have been studied by others. Klentzeris et al. (1995) showed that both macrophages and lymphocytes increased significantly between the early and late luteal phase of the menstrual cycle in both groups. Neither Jones et al. (1996) could show any quantitative difference in stromal leukocyte infiltration between the two groups. Braun et al. (2002), however, saw reduced numbers of macrophages during the early proliferative and late secretory phases in women with endometriosis, and correlated this to a reduced apoptotic activity during these phases. Thus, according to these studies, it is not likely that the clusters in our study represent single inflammatory cells, producing uPA or PAI-1.
uPAR mRNA expression was significantly higher in endometriotic glands and stromal cells and in endometrial glands from women with endometriosis compared to endometrial glands and stromal cells from controls. TGF-ß1 and TNF-, as well as EGF, induce uPAR mRNA expression in cancer cells (Blasi, 1993; Irigoyen et al., 1999). In colon carcinomas, the invading epithelial cells produce uPAR, while uPA is made by adjacent fibroblast-like stromal cells (Pyke et al., 1991). This may suggest a paracrine mechanism for the invading process with uPAR positive tumour cells inducing uPA from neighbouring cells and thus actively utilizing stromal products for invasion (Pyke et al., 1991). The same process could be proposed for uterine endometrium from women with endometriosis invading the peritoneal lining and endometriotic tissue invading underlying ECM. uPAR is also induced by nerve growth factor (NGF) in PC12 (pheochromocytoma) cells, and the importance of uPAR in NGF-driven differentiation of those cells has been studied by Farias-Eisner et al. (2000, 2001). The up-regulation of fibrinolytic factors might have consequences for the degradation of the endometrium during menstrual shedding, leading to tissue fragments with a higher potential of implantation onto peritoneal lining (Sillem et al, 1996). In endometriotic tissue, as in malignant tissue, the higher content of uPA, bound to its receptor uPAR, may contribute to the invasive growth by pericellular proteolysis and cell migration into surrounding tissue, and to metastasis to extragenital sites by cleaving plasminogen to plasmin (Danø et al., 1985; Andreasen et al., 2000).
Besides its protective capacity against uPA degradation of ECM, PAI-1 interferes with the binding of uPAR to vitronectin, a glycoprotein present in ECM and binding several integrins, and thus may potentially modulate cell adhesion and migration (Kjøller et al., 1997). PAI-1 may also promote tumour growth through inhibition of apoptosis (Kwaan et al., 2000). uPA binds to uPAR with high affinity. The most obvious consequence of elevated levels of uPA and uPAR is enhanced plasmin generation, leading to degradation of fibrin and other ECM proteins (Andreasen et al., 1997). Plasmin also catalyses activation of latent TGF-ß and the release of basic fibroblast growth factor (bFGF) from its ECM-binding sites, as well as contributing to activation of zymogens of MMP (Dumler et al., 1998). Taken together, this accounts for pericellular proteolysis and invasive properties with elevated levels of uPA protein and high levels of uPAR mRNA in endometriotic tissue.
Usually, plasminogen activation and plasmin generation leads to degradation of adhesion receptors and their ECM ligands, therefore counteracting cell–substratum and cell–cell adhesion. However, under certain conditions, binding of uPA to uPAR promotes cell–substratum adhesion. uPAR can act as an adhesion receptor, by uPA-stimulated binding of uPAR to vitronectin, and thereby regulate the functions of integrins, in an integrin-, ECM-, and cell type-specific manner (Andreasen et al., 2000). Adhesion of endometrial fragments in vitro to human peritoneum has been demonstrated by Groothuis et al. (1999). The elevated levels of uPA protein and high uPAR mRNA contents in endometrium from women with endometriosis may contribute to adhesion of shed endometrial fragments to peritoneal lining.
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Acknowledgements |
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We would like to thank Ass. Prof. Günther Weber for methodological guidance on transcription and linearization work, and Ass. Prof. Nils Lindefors for valuable discussions during in situ hybridization work. We also thank prof. Kjell Carlström for kindly helping us with statistical analyses. Financial support was provided by Anders Otto Swärd’s Foundation and by the Foundations of Golje and Lundströms Memorial.
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Submitted on October 9, 2003; accepted on December 4, 2003.
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