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ABSTRACT: 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.
Journal of Clinical Endocrinology & Metabolism 05/2001; 86(4):1724-30. · 6.50 Impact Factor
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ABSTRACT: 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.
Biology of Reproduction 11/1998; 59(4):759-67. · 4.01 Impact Factor
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ABSTRACT: The purpose of this study was to identify cyclic variations and hormonal regulation of endometrial transforming growth factor beta1 (TGFbeta1) mRNA. Regulation of the plasminogen-activating system was also examined, since it is involved in activation of latent TGFbetas. We measured TGFbeta1 mRNA in 51 normal endometrial samples by Northern blot and densitometric scanning of autoradiograms. Each value was related to the corresponding beta-actin value to allow quantitative evaluation. TGFbeta1 mRNA was higher in the mid and late secretory and menstrual phases than in the earlier parts of the cycle. This pattern implies progesterone dependence. The content of TGFbeta1 mRNA in endometrial tissue explants obtained in the proliferative phase was significantly increased after stimulation for 4 days with estradiol + progesterone in vitro. Both TGFbeta1 and estradiol + progesterone increased the content of plasminogen activator inhibitor-1 mRNA and protein in primary cultures of endometrial stromal cells. Conditioned-medium concentrations of urokinase plasminogen activator (u-PA) were increased by TGFbeta1, but decreased by estradiol + progesterone. This effect of estradiol + progesterone results from increased internalization and degradation of u-PA secondary to up-regulation of the cell surface receptor for u-PA by progesterone (Casslén et al., JCEM 1995; 80: 2776-2784). Increased extracellular u-PA in response to TGFbeta1 exposure was thus in concordance with an unchanged amount of available u-PA receptors on the cell surface. The activation mechanism of latent TGFbeta involves u-PA activity; since u-PA activity is reduced in the secretory endometrium, we suggest that although TGFbeta1 mRNA is increased in the mid and late secretory phase, TGFbetas are mainly in their latent form until the premenstrual rise in u-PA activity stimulates activation. TGFbeta may promote capillary growth during endometrial regeneration.
Biology of Reproduction 07/1998; 58(6):1343-50. · 4.01 Impact Factor
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ABSTRACT: Plasminogen activator inhibitor-1 (PAI-1) has important regulatory functions in haemostasis, extracellular matrix turn-over and cell adhesion. We studied PAI-1 gene expression in primary cultures of endometrial stromal cells, and found that PAI-1 protein and mRNA were increased both by agents associated with differentiation, i.e. progesterone and transforming growth factor beta1 (TGFbeta1), and by those promoting proliferation, i.e. epidermal growth factor (EGF), TGFalpha and basic fibroblast growth factor (bFGF). In order to further elucidate the mechanism of regulation, we transfected stromal cells with an expression construct containing 804 bp of the PAI-1 promoter fused to a chloramphenicol acetyl transferase (CAT) reporter gene. After stimulation with the polypeptide growth factors TGFbeta1, EGF and bFGF we found increased CAT activity, indicating that these stimulators had initiated interaction with the transfected promoter fragment. On the other hand, stimulation with progesterone did not increase CAT activity, even though these cells were perfectly able to respond with increased secretion of PAI-1 protein. Run off experiments demonstrated that progesterone increased the stability of PAI-1 mRNA in endometrial stromal cells. We conclude that the polypeptide growth factors TGFbeta1, EGF and bFGF increase PAI-1 expression by increasing gene transcription. Progesterone, on the other hand, does not interact with the 804 bp promoter region, but increases the stability of PAI-1 mRNA.
Molecular Human Reproduction 10/1997; 3(9):781-7. · 3.85 Impact Factor
