Differential effects of thrombin and hypoxia on endometrial stromal and glandular epithelial cell vascular endothelial growth factor expression

Ovarian steroids and/or premenstrual endometrial hypoxia are thought to restore the endometrial vasculature shed during menstruation by elevating endometrial vascular endothelial growth factor (VEGF) levels. During the luteal phase, VEGF levels peak, progesterone induces estradiol (E(2))-primed human endometrial stromal cells (HESCs) to decidualize and express tissue factor (TF), and endometrial vascular permeability is enhanced. The latter would present circulating clotting factors to decidual cell-expressed TF to form local thrombin. HESCs were incubated in serum-supplemented medium containing vehicle (control) or 10(-8) M E(2) or 10(-7) M medroxyprogesterone acetate (MPA) or E(2) + MPA for 7 d to induce decidualization, while monolayers of human endometrial glandular epithelial cells (HEGECs) formed during 4-d incubation of glands. The medium was exchanged for a defined medium containing corresponding vehicle or steroids +/- thrombin under normoxia or hypoxia (0-1% O(2)). Hypoxia enhanced secreted immunoreactive VEGF levels by severalfold in HESCs and HEGECs, but the steroids did not affect VEGF output in either cell type under normoxia or hypoxia. In E(2) + MPA-decidualized HESCs, VEGF levels were elevated by 0.1 U/ml of thrombin, and 0.5-2.5 U/ml of thrombin elicited maximum effects. The addition of 0.5 U/ml of thrombin evoked a time-dependent enhancement of VEGF levels and about an 8-fold increase at 48 h (P < 0.02; n = 6). Northern blotting indicated that E(2) + MPA-decidualized HESCs expressed VEGF(121), VEGF(165), and VEGF(189) mRNA, which were enhanced severalfold during 5- to 20-h incubation with thrombin. Moreover, TRAP, a synthetic peptide activator of the constitutively expressed protease activated receptor-1 thrombin receptor in decidualized HESCs, also elevated secreted VEGF levels. By contrast, HEGECs were unresponsive to thrombin added alone or with ovarian steroids. These results suggest that thrombin formed by progestin-augmented TF levels acts as an autocrine enhancer of VEGF expression in decidualized HESCs. Because angiogenesis occurs in a matrix of decidualized HESCs, these in vitro results provide a novel mechanism to account for both the peak in VEGF and angiogenesis in luteal phase human endometrium.     

Vascular endothelial growth factor expression in human endometrium is regulated by hypoxia

Endometrial growth and repair after menstruation are associated with profound angiogenesis. Abnormalities in these processes result in excessive or unpredictable bleeding patterns and are common in many women. It is therefore important to understand which factors regulate normal endometrial angiogenesis. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that plays an important role in normal and pathological angiogenesis. In this study we show that expression of VEGF is regulated by hypoxia in human endometrium. Culture in vitro for 24 h under hypoxic conditions resulted in a 2- to 6-fold increase in VEGF secretion by both stromal and epithelial cells isolated from human endometrium. Quantitative RT-PCR was used to measure VEGF messenger ribonucleic acid (mRNA) levels in these cells. After hypoxia, VEGF mRNA levels increased 1.8-fold in stromal cells and 3.4-fold in glandular epithelial cells. The mRNA for each VEGF splice variant increased to an equal extent. The increase in VEGF secretion by stromal and epithelial cells in response to hypoxia was not altered by treatment at the same time with estradiol or progesterone. In situ hybridization of human endometrium during menstruation, when steroid levels are low but the tissue is subject to ischemia, showed strong hybridization to VEGF mRNA in both stromal and glandular cells. These results show that local factors, such as hypoxia, can regulate VEGF expression in the endometrium. This may play an important part in normal endometrial repair after menstruation. The secretion of VEGF by endometrial cells under hypoxic conditions may also be important in the pathogenesis of endometriosis, because it would be predicted to assist revascularization of desquamated endometrial explants when they attach at ectopic sites.     

Dickkopf-1, an inhibitor of Wnt signaling, is regulated by progesterone in human endometrial stromal cells

CONTEXT: Some members of the Wnt family, including ligands, receptors, inhibitors, and signaling components, are expressed in human endometrium. Dickkopf-1 (Dkk-1), a potent inhibitor of the Wnt signaling pathway, was recently found to be up-regulated in decidualizing endometrial stromal cells during the secretory phase of the menstrual cycle, suggesting regulation by progesterone. OBJECTIVES: To test the hypothesis that progesterone regulates Dkk-1 expression in human endometrial stromal cells, we investigated the following effects on stromal cell expression of Dkk-1 mRNA and protein: decidualizing stimuli (progesterone or cAMP), RU486 (an inhibitor of progesterone action), and withdrawal of progesterone. RESULTS: Short-term treatment (up to 72 h, which corresponds to the full decidualized phenotype in response to cAMP and an early response to progesterone) did not reveal regulation of Dkk-1 mRNA or protein by cAMP but did show induction of Dkk-1 expression when the cells were treated with progesterone, an effect that was blocked by RU486. In long-term cultures (from 14 to 23 d, which corresponds to the full decidualized phenotype in response to progesterone), a significant increase in Dkk-1 mRNA and protein production was observed. Addition of RU486 or withdrawal of progesterone after long-term decidualization resulted in a decrease of Dkk-1 mRNA and protein to control levels. Estradiol alone had no effect on stromal Dkk-1 expression. CONCLUSIONS: These data strongly support regulation by progesterone of Dkk-1 mRNA synthesis and protein expression in human endometrial stromal cells and that the response is specific for progesterone and independent of cAMP and estradiol.     

Demonstration of angiogenin in human endometrium and its enhanced expression in endometrial tissues in the secretory phase and the decidua.

Angiogenesis is thought to be crucial for normal physiology of the endometrium, where dynamic vascular remodeling occurs during the menstrual cycle and pregnancy. We investigated the presence of angiogenin, a potent inducer of angiogenesis, and the regulatory mechanisms of its production in the human endometrium. Western blot analysis demonstrated that angiogenin protein expression increased by 3- to 4-fold in the endometrium in the mid and late secretory phases and in early gestation relative to that during the proliferative phase. Quantitative mRNA analysis showed the similar tendency in the expression of angiogenin mRNA in the endometrium, with the highest levels observed in the mid and late secretory phases and early gestation. An immunohistochemical study showed that angiogenin was expressed in both stromal cells and epithelial cells, with indistinguishable intensity between these cells regardless of phases of the menstrual cycle. In support of the Western blot analysis, the intensity of staining appeared to be highest in the mid to late secretory phases relative to other phases. Consistent with these in vivo results, decidualized cultured stromal cells, after treatment with progesterone or progesterone plus E2, exhibited the capacity to secrete significantly increased amounts of angiogenin compared with untreated or E2 alone-treated control group. Both the treatment with (Bu)2cAMP and hypoxic conditions stimulated angiogenin secretion by stromal cells. For isolated epithelial cells, hypoxia stimulated angiogenin secretion, whereas (Bu)2cAMP had no appreciable effect. In summary, we demonstrated the presence of angiogenin in human endometrium and its possible local regulatory factors, such as progesterone, cAMP, and hypoxia. These findings along with its enhanced expression in the endometrium in the secretory phase and in decidual tissues raise the possibility that angiogenin may play a role in establishing pregnancy.     

Wild-type p53 protein is up-regulated upon cyclic adenosine monophosphate-induced differentiation of human endometrial stromal cells

Decidualization of the endometrial stromal compartment is critical for embryo implantation. Initiation of this differentiation process requires elevated intracellular cAMP levels. We now report a massive and sustained up-regulation of p53 tumor suppressor protein during cAMP-induced decidualization of cultured endometrial stromal cells. Nuclear accumulation of p53 was not accompanied by increased mRNA expression, suggesting stabilization of the protein as the underlying mechanism. Proteasomal degradation of p53 is known to be mediated by nuclear Mdm2. Nuclear translocation of Mdm2, in turn, is dependent on phosphorylation by protein kinase B/Akt (PKB/Akt). In cAMP-treated decidualized cells, p53 accumulation was associated with decreased nuclear Mdm2 and cytoplasmic PKB/Akt levels. Conversely, withdrawal of the decidualization stimulus resulted in morphological and biochemical dedifferentiation, disappearance of p53, but increased abundance of PKB/Akt. Furthermore, Western blot and immunohistochemical analyses of endometrial biopsies confirmed that p53 is expressed in vivo in the stromal compartment during the late secretory phase of the cycle. The observation that p53 protein expression is closely associated with decidual transformation indicates a novel role for this tumor suppressor in regulating human endometrial function.     

Effects of thrombin, hypoxia, and steroids on interleukin-8 expression in decidualized human endometrial stromal cells: implications for long-term progestin-only contraceptive-induced bleeding

Abnormal uterine bleeding is the major reason for discontinuing long-term progesterone-only contraceptives (LTPOCs). Prior studies demonstrated that endometria exposed to the LTPOC, Norplant, display aberrant angiogenesis, leukocyte infiltration, and hypoxia-associated impaired blood flow. Paradoxically, human endometrial stromal cells (HESCs) of these specimens exhibit elevated expression of tissue factor (TF), the primary initiator of hemostasis via thrombin generation. The current study demonstrates that TF levels are also elevated in HESCs that are decidualized after insertion of Mirena, an intrauterine system that releases levonorgestrel directly into the endometrial canal and produces elevated perivascular levels of the proinflammatory and angiognenic cytokine IL-8. Because bleeding, inflammation, and ischemia-associated increased vascular permeability enhance access of plasma factor VII to HESC-expressed TF to generate thrombin, we evaluated the effects of steroids, thrombin, and hypoxia on HESC expression of IL-8. Confluent HESCs were incubated in a serum-containing medium for 7 d with vehicle control or estradiol (E(2)) plus medroxyprogesterone acetate (MPA). The medium was then exchanged for corresponding defined medium with and without thrombin, and the cultures were incubated in parallel for up to 48 h in a standard incubator (normoxia) or a sealed chamber at 0-1% O(2) (hypoxia). Under normoxia, immunoreactive IL-8 levels in the conditioned medium were reduced to one-third of control levels during decidualization with E(2)+MPA (P < 0.05; n = 5). In E(2)+MPA-treated cultures, thrombin (0.1 U/ml to 2.5 U/m) elicited a dose-dependent reversal of this inhibition, elevating IL-8 up to 60-fold (P < 0.05; n = 5) for more than 24 h and steady-state IL-8 mRNA levels by 3-fold for 3 h. The specific inactivator, hirudin, blocked most of the effects of thrombin, whereas TRAP-14, an agonist of the protease-activated receptor for thrombin, enhanced IL-8 output. In the absence of thrombin, hypoxia elevated IL-8 output 5-fold in E(2)+MPA-treated HESCs (P < 0.02, n = 4), with thrombin exerting additive effects. In contrast to its effects in progestin-treated HESCs, hypoxia did not elevate IL-8 output in control cultures. This study suggests that inhibition of IL-8 expression in decidualized HESCs contributes to the antiinflammatory milieu of the luteal phase. However, LTPOC-induced hypoxia and excess thrombin generation enhance IL-8 expression in decidualized HESCs, thereby eliciting aberrant angiogenesis and inflammation that promote the onset of abnormal uterine bleeding.     

Relaxin and prostaglandin E(2) regulate interleukin 11 during human endometrial stromal cell decidualization

Decidualization of endometrial stromal cells and IL-11 signaling are essential for embryo implantation in the mouse. We investigated the effects of relaxin (RLX) and prostaglandin E(2) (PGE(2)) on IL-11 secretion by human endometrial stromal cells (HESC) and during cAMP or medroxyprogesterone acetate (P)-induced decidualization. cAMP-decidualized HESC secreted high levels of IL-11. RLX, cAMP, or PGE(2) increased IL-11 mRNA and IL-11 secretion, with maximal response to RLX and cAMP. Addition of the cAMP/protein kinase A inhibitor Rp-adenosine-3,5-cyclic-monophosphorothioate to either RLX- or PGE(2)-treated cells decreased IL-11 secretion. Indomethacin treatment decreased IL-11 secretion, which was largely restored by cotreatment with PGE(2) or RLX. Cotreatment of HESC with RLX, PGE(2), or cAMP and estrogen plus P down-regulated IL-11 mRNA and IL-11 secretion at 24 h, before secretion of prolactin (decidualization marker). Addition of W147AIL-11 (IL-11 signaling inhibitor) reduced prolactin secretion stimulated by RLX or PGE(2) and estrogen plus P. This is the first demonstration that cAMP-decidualized HESC secrete IL-11 and that IL-11 mRNA and IL-11 secretion are regulated by RLX and PGE(2), partly via a cAMP/protein kinase A-dependent pathway. Blocking IL-11 signaling reduced RLX+P- or PGE(2)+P-induced decidualization, suggesting that RLX and PGE(2) act via IL-11. This is important in understanding implantation and regulation of fertility.     

Identification and regulation of the IGFBP-4 protease and its physiological inhibitor in human trophoblasts and endometrial stroma: evidence for paracrine regulation of IGF-II bioavailability in the placental bed during human implantation

The IGF family plays an important role in implantation and placental physiology. IGF-II is abundantly expressed by placental trophoblasts, and IGF binding protein (IGFBP)-4, a potent inhibitor of IGF actions, is the second most abundant IGFBP in the placental bed, expressed exclusively by the maternal decidua. Proteolysis of IGFBP-4 results in decreased affinity for IGF peptides, thereby enhancing IGF actions. In the current study, we have identified the IGFBP-4 protease and its inhibitor in human trophoblast and decidualized endometrial stromal cell cultures, and we have investigated their regulation in an effort to understand control of IGF-II bioavailability at the placental-decidual interface in human implantation. IGFBP-4 protease activity was detected in conditioned media (CM) from human trophoblasts and decidualized endometrial stromal cells using (125)I-IGFBP-4 substrate. Identification of the IGFBP-4 protease as pregnancy-associated plasma protein-A (PAPP-A) was confirmed by specific immunoinhibition and immunodepletion of the IGFBP-4 protease activity with specific PAPP-A antibodies. The IGFBP-4 protease activity was IGF-II-dependent in trophoblast CM. In decidualized stromal CM, PAPP-A/IGFBP-4 protease activity was also IGF-II-dependent, but was evident only when IGF-II was added in molar excess of the predominant IGFBP in decidualized stromal cell CM, IGFBP-1, supporting bioavailable IGF-II as a key cofactor of IGFBP-4 proteolysis by PAPP-A. Cultured first and second trimester human trophoblasts (n = 5) secreted PAPP-A into CM with mean +/- SEM levels of 172.4 +/- 32.8 mIU/liter.10(5) cells, determined by specific ELISA. PAPP-A in trophoblast CM (n = 3) and did not change in the presence of IGF-II (1-100 ng/ml). Cultured human endometrial stromal cells (n = 4) secreted low levels of PAPP-A (6.25 +/- 3.6 mIU/liter.10(5) cells). A physiological inhibitor of PAPP-A, the proform of eosinophil major basic protein (proMBP), was detected in trophoblast CM at levels of 1853 +/- 308 mIU/liter.10(5) cells, determined by specific ELISA, and was nearly undetectable in CM of human endometrial stromal cells. Upon in vitro decidualization of endometrial stromal cells with progesterone, PAPP-A levels in CM increased nearly 9-fold without a concomitant change in proMBP. In contrast to the experiments with trophoblasts, IGF-II and the IGF analogues, Leu(27) IGF-II, and Des (1-6) IGF-II, resulted in a dose-dependent decrease of PAPP-A levels in decidualized endometrial stromal CM by 70-90%, and a dose-dependent increase in proMBP of 14- to 41-fold. The data demonstrate conclusively that the IGF-II-dependent IGFBP-4 protease of human trophoblast and decidual origin is PAPP-A. Furthermore, the differential regulation of decidual PAPP-A and proMBP by insulin-like peptides supports a role for trophoblast-derived IGF-II as a paracrine regulator of these maternal decidual products that have the potential to regulate IGF-II bioavailability at the trophoblast-decidual interface. Overall, the data underscore potential roles for a complex family of enzyme (PAPP-A), substrate (IGFBP-4), inhibitor (proMBP), and cofactor (IGF-II) in the placental bed during human implantation.     

Regulation of the production of placental protein 5 by human endometrial stromal cells; the role of prostaglandins E2 and F2 alpha

Cultured human endometrial stromal cells were found to release placental protein 5 (PP5), a glycoprotein with properties of a serine protease inhibitor. Progesterone had no effect on PP5 release, but cholera toxin and 12-O-tetradecanoylphorbol 13-acetate stimulated PP5 release in a time- and concentration-dependent fashion. Prostaglandin E2 (PGE2) caused a parallel increase in cAMP and PP5 release in a time- and concentration-dependent fashion. The lowest PGE2 concentration which increased cAMP and PP5 release was 1 X 10(-9) M. Maximal increase in cAMP (42-fold) and PP5 (25-fold) release was obtained by 10(-5) M PGE2. Stimulation of cAMP by PGE2 was detectable at 15 min and was followed by an increased PP5 release at 24 h. The concentrations of prostaglandin F2 alpha (PGF2 alpha) which stimulated cAMP and PP5 release were pharmacological suggesting that this effect is nonspecific. The results indicate that the activation of cAMP- and protein kinase C-dependent pathways in endometrial stromal cells increases the production of PP5. PGE2 could be one of the physiological ligands employing the cAMP-dependent pathway in endometrial stromal cells.     

Dose-dependent insulin regulation of insulin-like growth factor binding protein-1 in human endometrial stromal cells is mediated by distinct signaling pathways

IGF binding protein-1 (IGFBP-1) is a major product of decidualized human endometrial stromal cells and decidua, and as a modulator of IGF action and/or by independent mechanisms, it regulates cell growth and differentiation and embryonic implantation in these tissues. IGFBP-1 secretion is primarily stimulated by progesterone and cAMP and is inhibited by insulin and IGFs. The signaling pathways mediating the latter are not well defined, and the current study was conducted to determine which pathways mediate the effects of insulin on IGFBP-1 mRNA and protein expression by human endometrial stromal cells decidualized in vitro by progesterone. Cells were cultured and treated with different combinations of insulin; wortmannin, an inhibitor of the phosphatidylinositide-3-kinase (PI3-kinase) pathway; and PD98059, an inhibitor of the MAPK pathway. IGFBP-1 mRNA was determined by real-time PCR, and protein secretion in the conditioned medium was measured by ELISA. Activation of the PI3-kinase and the MAPK pathways was assessed by the detection of phosphorylated AKT and ERK in Western blots, respectively. Insulin inhibited IGFBP-1 mRNA and protein secretion in a dose-dependent fashion, with an ED(50) for the latter 0.127 ng/ml (21.6 pm). Inhibitor studies revealed that at low doses, insulin acts through the PI3-kinase pathway, whereas at higher levels it also activates the MAPK pathway in the inhibition of IGFBP-1. The data demonstrate that human endometrium is a target for insulin action in the regulation of IGFBP-1. At physiological levels insulin likely plays a homeostatic role for energy metabolism in the endometrium, and in hyperinsulinemic states, insulin action on the endometrium may activate cellular mitosis via the MAPK pathway and perhaps predispose this tissue to hyperplasia and/or cancer.     

Steroid and peptide regulation of insulin-like growth factor-binding proteins secreted by human endometrial stromal cells is dependent on stromal differentiation.

Endometrial stromal cells undergo decidual transformation, in response to epidermal growth factor (EGF) and progesterone. Since insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) are believed to be involved in endometrial differentiation, and insulin regulates IGFBP production in a variety of cells, we have investigated the modulatory roles of EGF, progesterone, and insulin on IGFBP secretion by long term cultures of human endometrial stromal cells. Without insulin, the principal IGFBP secreted into conditioned medium, detected by Western ligand blotting, was a 28-kilodalton (kDa) IGFBP, identified by immunoprecipitation as IGFBP-1. This was observed only when the stromal cells were decidualized. With increasing insulin, IGFBP-1 decreased to undetectable levels. Concomitantly, IGFBP-2 increased, as did a 24-kDa IGFBP (believed to be IGFBP-4) and a 28-kDa IGFBP, shown to be a glycoprotein by endoglycosidase sensitivity (and believed to be glycosylated IGFBP-4). In the nondecidualized state, insulin increased the secretion of IGFBP-3, IGFBP-2, and the 24-kDa IGFBP, which were slightly inhibited by EGF and relatively unaffected by progesterone alone. In the absence of insulin, progesterone weakly stimulated IGFBP-1 secretion, which increased markedly when the cells were decidualized by combined treatment with EGF and progesterone. These data show that IGFBP-3, IGFBP-2, IGFBP-1, and presumably IGFBP-4 and its glycosylated form are differentially regulated by peptide and steroid hormones in endometrial stromal cells and that their regulation is a function of stromal differentiation.     

Assessment of VEGF-receptor system expression in the porcine endometrial stromal cells in response to insulin-like growth factor-I, relaxin, oxytocin and prostaglandin E2

Several factors participate in regulation of growth and development as well as angiogenesis of the uterus during pregnancy, and hence little is known about the role of hormonal regulation of vascular endothelial growth factor (VEGF)-receptor system expression. This study has examined the effect of insulin-like growth factor-I (IGF-I), relaxin (RLX), oxytocin (OT) and prostaglandin (PG) E(2), on VEGF secretion and VEGF-receptor system mRNA expression in the porcine endometrial stromal cells. IGF-I and RLX were identified as the most effective inducers of VEGF secretion and mRNA expression. Although PGE(2) stimulated VEGF secretion and VEGF164 mRNA expression, OT inhibited both secretion and mRNA expression of VEGF. When tested for VEGF receptors (R), all factors failed to affect their mRNA expression. Media conditioned by stromal cells collected after IGF-I and RLX treatment significantly increased endothelial cell proliferation and this effect was blocked by soluble VEGFR-1. These data suggest that during early pregnancy IGF-I, RLX and PGE(2) can affect VEGF expression in the endometrium and therefore may support uterine and embryo development, implantation and pregnancy.     

Vascular endothelial growth factor mediates the estrogen-induced breakdown of tight junctions between and increase in proliferation of microvessel endothelial cells in the baboon endometrium

To assess whether there is a link between estrogen, vascular endothelial growth factor (VEGF), and early aspects of uterine angiogenesis, an acute temporal study was conducted in which ovariectomized baboons were pretreated with VEGF Trap, which sequesters endogenous VEGF, and administered estradiol at time 0 h. Serum estradiol levels approximated 500 pg/ml 4-6 h after estradiol administration. VEGF mRNA levels in endometrial glandular epithelial and stromal cells were increased to values 6 h after estradiol that were 3.74 +/- 0.99-fold (mean +/- se) and 5.70 +/- 1.60-fold greater (P < 0.05), respectively, than at 0 h. Microvessel interendothelial cell tight junctions, which control paracellular permeability, were present in the endometrium at time 0 h, but not evident 6 h after estradiol administration. Thus, microvessel paracellular cleft width increased (P < 0.01, ANOVA) from 5.03 +/- 0.22 nm at 0 h to 7.27 +/- 0.48 nm 6 h after estrogen. In contrast, tight junctions remained intact, and paracellular cleft widths were unaltered in estradiol/VEGF Trap and vehicle-treated animals. Endometrial microvessel endothelial cell mitosis, i.e. percent Ki67+/Ki67- immunolabeled endothelial cells, increased (P < 0.05) from 2.9 +/- 0.3% at 0 h to 21.4 +/- 7.0% 6 h after estrogen treatment but was unchanged in estradiol/VEGF Trap and vehicle-treated animals. In summary, the estrogen-induced disruption of endometrial microvessel endothelial tight junctions and increase in endothelial cell proliferation were prevented by VEGF Trap. Therefore, we propose that VEGF mediates the estrogen-induced increase in microvessel permeability and endothelial cell proliferation as early steps in angiogenesis in the primate endometrium.