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ABSTRACT: Invasion of the decidua by extravillous trophoblasts (EVTs) is accompanied by thrombin generation from decidual cell (DC)-expressed tissue factor (TF). This TF protects against hemorrhage as EVTs breach capillaries and subsequently invade and remodel spiral arteries and arterioles. Pre-eclampsia (P-EC) is the world's leading cause of fetal and maternal morbidity and mortality. It is associated with decidual hemorrhage and maternal thrombophilias, which form excess thrombin from DCs, and with maternal infections and other inflammatory conditions that are associated with excess expression of the proinflammatory cytokines interleukin (IL)-1 β and tumor necrosis factor (TNF) α. In human first-trimester leukocyte-free DCs, (1) thrombin enhances expression of soluble fms-like tyrosine kinase-1 (sFlt-1), a potent inhibitor of angiogenesis; (2) thrombin, IL-1β and TNF-α increase monocyte-recruiting chemokine expression leading to a macrophage excess in the pre-eclamptic decidua. The pathogenesis of P-EC likely stems from shallow EVT invasion leading to impaired decidual vascular remodeling. The resulting reduced uteroplacental blood flow is associated with a hypoxic placenta, which appears to secrete excess sFlt-1 into the maternal plasma. A regulatory role for DCs in vascular remodeling is indicated because impaired decidual vascular remodeling could stem from an aberrant local antiangiogenic milieu elicited by excess sFlt-1 and/or macrophage-inhibited EVT decidual invasion.
Seminars in Thrombosis and Hemostasis 03/2011; 37(2):158-64. · 4.52 Impact Factor
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ABSTRACT: Abnormal uterine bleeding is the leading indication for discontinuation of long-term progestin-only contraceptives. Histological sections of endometria from long-term progestin-treated patients display abnormally enlarged blood vessels that are prone to bleed in a nonuniform manner. Because it has been complex to attain patients willing to complete long-term studies, and good quality endometrial tissues have proven difficult to obtain, animal models have been used to obviate this problem. In this review, we describe these models including the guinea pig, an animal model we have previously investigated, to assess the mechanisms involved in abnormal uterine bleeding following long-term progestin-only contraception.
Annals of the New York Academy of Sciences 03/2011; 1221:119-23. · 3.15 Impact Factor
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S Joseph Huang,
Ana C Zenclussen,
Chie-Pein Chen,
Murat Basar,
Hui Yang,
Felice Arcuri,
Min Li,
Erdogan Kocamaz,
Lynn Buchwalder, Mizanur Rahman,
Umit Kayisli,
Frederick Schatz,
Paolo Toti,
Charles J Lockwood
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ABSTRACT: Preeclampsia is characterized by an exaggerated systemic inflammatory state as well as shallow placentation. In the decidual implantation site, preeclampsia is accompanied by an excessive number of both macrophages and dendritic cells as well as their recruiting chemokines, which have been implicated in the impairment of endovascular trophoblast invasion. Granulocyte-macrophage colony-stimulating factor is known to regulate the differentiation of both macrophages and dendritic cells, prompting both in vivo and in vitro evaluation of granulocyte-macrophage colony-stimulating factor expression in human decidua as well as in a mouse model of preeclampsia. This study revealed increased granulocyte-macrophage colony-stimulating factor expression levels in preeclamptic decidua. Moreover, both tumor necrosis factor-α and interleukin-1 β, cytokines that are implicated in the genesis of preeclampsia, markedly up-regulated granulocyte-macrophage colony-stimulating factor production in cultured first-trimester human decidual cells. The conditioned media of these cultures promoted the differentiation of both macrophages and dendritic cells from a monocyte precursor. Evaluation of a murine model of preeclampsia revealed that the decidua of affected animals displayed higher levels of immunoreactive granulocyte-macrophage colony-stimulating factor as well as increased numbers of both macrophages and dendritic cells when compared to control animals. Because granulocyte-macrophage colony-stimulating factor is a potent inducer of differentiation and activation of both macrophages and dendritic cells, these findings suggest that this factor plays a crucial role in the pathogenesis of preeclampsia.
American Journal Of Pathology 11/2010; 177(5):2472-82. · 4.89 Impact Factor
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ABSTRACT: During extravascular trophoblast (EVT) invasion of the decidua, thrombin generated from decidual cell-expressed tissue factor (TF) forms a "hemostatic envelope" that protects against hemorrhage during the initial breaching of capillaries by EVTs and subsequent invasion and remodeling of the spiral arteries and arterioles. Preeclampsia, the world's leading cause of fetal and maternal morbidity and mortality, stems from shallow trophoblast invasion leading to incomplete vascular remodeling that impairs uteroplacental blood flow. A considerable subset of cases of preeclampsia is associated with decidual hemorrhage and maternal thrombophilias, which form excess thrombin from decidual cell-expressed TF. Thrombin affects several cell functions by binding to protease-activated receptors. In first-trimester decidual cells, thrombin enhances expression of sFlt-1, which can block the angiogenic effects of vascular endothelial growth factor (VEGF) and placental growth factor. By contrast, thrombin does not affect decidual cell VEGF expression. Thrombin-enhanced sFlt-1 expression by decidual cells, the predominant cell type encountered by invading cytotrophoblasts, could promote preeclampsia by interfering with angiogenesis-dependent vascular remodeling to reduce uteroplacental blood flow.
Annals of the New York Academy of Sciences 05/2008; 1127:67-72. · 3.15 Impact Factor
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ABSTRACT: Excess decidual macrophage infiltration has been linked to preeclampsia and a failure of endovascular trophoblast invasion. Severe preeclampsia with shallow placentation has also been linked to acquired and inherited maternal thrombophilias and recurrent decidual hemorrhage, which generates thrombin from decidual cell-expressed tissue factor. Therefore, the current study evaluated whether thrombin affects monocyte chemoattractant protein-1 (MCP-1) expression in stromal cells that are derived from cycling and gestational endometrium.
Stromal cells that are isolated from cycling endometrium and first trimester and term decidua were grown to confluence, treated for 7 days with 10(-8) mol/L estradiol (E2) + 10(-7) mol/L medroxyprogesterone acetate (MPA), then switched to a serum-free medium that contained the corresponding steroids +/- thrombin. MCP-1 protein release was measured by enzyme-linked immunosorbent assay and Western blot; MCP-1 messenger RNA levels were assessed by real-time quantitative reverse transcription-polymerase chain reaction.
Secreted MCP-1 levels were not significantly different in stromal or decidual cell cultures that were incubated with E2 or with E2 + MPA. Thrombin increased immunoreactive MCP-1 expression in a dose-response fashion in first trimester and term decidual cells but not in endometrial stromal cells. Thrombin-induced MCP-1 protein output was unaffected by MPA but was abrogated by incubation with the thrombin inhibitor, hirudin. Unexpectedly, thrombin-enhanced MCP-1 protein expression was unaccompanied by corresponding changes in steady state MCP-1 messenger RNA levels, which suggests its effects were posttranslational.
MCP-1 protein expression is up regulated by thrombin in decidual cells across gestation, but not in stromal cells from predecidualized cycling endometrium.
American journal of obstetrics and gynecology 04/2007; 196(3):268.e1-8. · 3.28 Impact Factor
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Errol R Norwitz,
Victoria Snegovskikh,
Frederick Schatz,
Nastaran Foyouzi, Mizanur Rahman,
Lynn Buchwalder,
Hee Joong Lee,
Edmund F Funai,
Catalin S Buhimschi,
Irina A Buhimschi,
Charles J Lockwood
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ABSTRACT: Labor is associated with 'decidual activation' with increased proteolysis and extracellular matrix degradation. The balance between plasminogen activator inhibitor-1 (PAI-1) and urokinase (uPA) and tissue-type plasminogen activator (tPA) is an important determinant of proteolytic activity at the maternal-fetal interface. Thrombin released at the time of placental abruption (decidual hemorrhage) is known to promote decidual proteolysis and uterine contractions. This study investigates the separate and interactive effects of steroid hormones and thrombin on PAI-1, uPA, and tPA expression by term decidual cells (DCs).
Term DCs were isolated by enzymatic digestion, purified, and depleted of leukocytes. Cells were treated with estradiol (10(-8) mol [E2]), medroxyprogesterone acetate (10(-7) mol [MPA]), both, or vehicle for 7 days. After 24-hour incubation with or without thrombin (0.1-2.5 U/mL), levels of PAI-1, uPA, and tPA in conditioned supernatant were measured by specific ELISA and Western blotting. Levels of PAI-1 and uPA mRNA were measured by quantitative RT-PCR.
In the cultured term DCs, ELISA measurements indicated that basal output of PAI-1 was about 2 logs higher than that of either uPA or tPA (2.5 +/- 0.7 ng/mL per microg protein, 13.4 +/- 6.3 pg/mL per microg protein, and 25.4 +/- 10.8 pg/mL per microg protein, respectively). Although E2 alone did not affect PAI-1 output, MPA and E2+MPA significantly enhanced PAI-1 production (2.5 +/- 0.7 vs 8.2 +/- 2.0 ng/mL per microg protein for E2+MPA [3.3-fold]; P < .01). By contrast, uPA output was inhibited by exposure to MPA (13.4 +/- 6.3 vs 2.6 +/- 1.1 pg/mL per microg protein [0.2-fold]; P < .05), whereas tPA production was not affected by MPA. Thrombin did not significantly affect uPA and tPA production by term DCs. In contrast, in E2+MPA-treated term DCs, thrombin, a hemostatic proinflammatory cytokine, selectively increased PAI-1 output in a dose-dependent fashion, which could be blocked by the selective thrombin inhibitor, hirudin. Western blotting confirmed the effects of MPA and thrombin in elevating secreted levels of PAI-1. Unlike the increase in PAI-1 output elicited by thrombin, term DCs were unresponsive to either of the classic proinflammatory cytokines, TNFalpha or IL-1beta. Corresponding effects on PAI-1 mRNA levels were elicited by MPA and thrombin as seen for PAI-1 protein expression, suggesting that these up-regulatory effects are transcriptionally mediated.
Progestin enhanced PAI-1 and inhibited uPA expression by term DCs, which may explain in part the pregnancy-prolonging properties of progesterone as a consequence of inhibited proteolytic activity at the maternal-fetal interface. Thrombin augmented PAI-1 expression in the absence of increased uPA or tPA expression by term DCs, suggesting that abruption-associated decidual proteolysis and preterm labor is mediated primarily by thrombin-enhanced matrix metalloproteinase expression rather than an indirect effect on the plasminogen activator/inhibitor system.
American journal of obstetrics and gynecology 04/2007; 196(4):382.e1-8. · 3.28 Impact Factor
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ABSTRACT: Progesterone-induced decidualized human endometrial stromal cells form a hemostatic envelope that protects against hemorrhage during invasion of endometrial capillaries by implanting blastocyst-derived cytotrophoblasts (CTs). This hemostatic milieu reflects co-upregulated expression of tissue factor (TF), the primary initiator of hemostasis via thrombin generation and plasminogen activator inhibitor type 1, which inactivates tissue-type plasminogen activator, the primary fibrinolytic agent. During deep invasion of the decidua, CTs breach and remodel spiral arteries and arterioles to produce high-conductance vessels. Shallow invasion results in incomplete vascular transformation and an underperfused fetal - placental unit associated with preeclampsia and intrauterine growth restriction. Decidual hemorrhage and severe thrombophilias elicit aberrant thrombin generation from decidual cell-expressed TF. Such thrombin induces decidual cells to synthesize and secrete soluble fms-like tyrosine kinase-1 (sFlt-1), the matrix metalloproteinases MMP-1 and MMP-3, and the neutrophil chemoattractant interleukin-8. Excess sFlt-1 at the implantation site may inhibit CT invasion by altering the angiogenic factor balance. During abruptions, thrombin-enhanced MMP-1, MMP-3 by decidual cells and neutrophil-derived proteases degrade the decidual and fetal membrane extracellular matrix to promote preterm premature rupture of the membranes. In association with long-term progestin-only contraception, overexpression of decidual cell-derived thrombin promotes aberrant angiogenesis and vessel maintenance to contribute to abnormal uterine bleeding.
Seminars in Thrombosis and Hemostasis 03/2007; 33(1):111-7. · 4.52 Impact Factor
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ABSTRACT: Tibolone and its metabolites were evaluated on matrix metalloproteinase (MMP) expression in human endometrial stromal cells (HESCs) under the hypothesis that these steroids would act as progestins on MMP-1, -2, and -3 expression. After 7 days of priming and 24h experimental incubation of confluent cultured HESCs, 10(-7) M medroxyprogesterone acetate (P) reduced MMP-1 to 49+/-34% (p<0.05) and MMP-3 to 33+/-22% of basal levels (mean+/-S.E.M., p<0.05, n=5). Although HESCs were unaffected by 10(-8) M estradiol (E), E+P reduced MMP-1 and MMP-3 levels an additional 2.5-fold from P alone. Tibolone and Delta-4 tibolone were equivalent to E+P in inhibiting MMP-1 and MMP-3 output, whereas 10(-6)M of 3alpha-OH or 3beta-OH tibolone was required to elicit significant inhibition of both MMPs (p<0.05). By contrast, none of the treatments affected HESC-secreted MMP-2 output. The ELISA results were confirmed by Western blotting and by substrate gel zymography. Quantitative RT-PCR demonstrated corresponding changes in MMP-1 and MMP-3 mRNA levels. Inhibition of MMP-1 and MMP-3 expression by tibolone and Delta-4 tibolone is consistent with the metabolism of tibolone to Delta-4 tibolone, and subsequent binding of Delta-4 tibolone to the progesterone receptor. Since 3alpha-OH and 3beta-OH tibolone bind exclusively to the estrogen receptor, their inhibition of MMP-1 and MMP-3 suggests metabolism by HESCs to Delta-4 tibolone. These observations help to explain the paradox that the endometrium becomes atrophic after tibolone administration despite the persistence in the circulation of 3alpha-OH and 3beta-OH tibolone, but not tibolone or Delta-4 tibolone.
Steroids 09/2006; 71(9):768-75. · 2.83 Impact Factor
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ABSTRACT: Decidual inflammation and hemorrhage are major contributors to the pathogenesis of preterm delivery (PTD). IL-11 is a cytokine with pleiotropic biological effects, including induction of T helper cell type 2 and inhibition of T helper cell type 1 cytokine responses. Paradoxically, it enhances the synthesis of prostaglandins, which induce labor.
The objectives of this study were to evaluate in vivo IL-11 expression in decidua after term and preterm deliveries and evaluate the effects of the primary mediators of inflammation, IL-1beta and TNF-alpha, as well as the primary regulator of hemostasis, thrombin, on IL-11 expression in cultured term decidual cells (DCs).
Human decidua from uncomplicated term deliveries and chorioamnionitis- or placental abruption-related PTDs were immunostained for IL-11. Cultures of DCs were primed with estradiol (E2) or with E2 and medroxyprogesterone acetate (MPA), then incubated in a defined medium with corresponding steroid(s) with or without IL-1beta, TNF-alpha, or thrombin. IL-11 levels in DC-defined media were assessed by ELISA and Western blotting; IL-11 mRNA levels were measured by quantitative RT-PCR.
IL-11 immunostaining was significantly higher in DCs after PTD compared with those after term delivery (P < 0.05). In the absence of cytokines or thrombin, IL-11 levels in the defined medium were 47% lower in incubations with E2 plus MPA vs. E2 alone (P = 0.001). IL-1beta and thrombin elevated IL-11 output during incubations with E2 [24-fold (P < 0.05) and 120-fold (P < 0.05), respectively]. These increases were blunted by the addition of MPA [13-fold (P < 0.05) and 36-fold (P < 0.05), respectively]. Western blot analysis confirmed the ELISA results, and RT-PCR demonstrated corresponding effects on IL-11 mRNA levels. Unexpectedly, TNF-alpha did not affect IL-11 levels.
Because excess IL-1beta and thrombin generation are associated with chorioamnionitis- and abruption-related PTD, respectively, these findings add to our understanding of the genesis of inflammation- and abruption-associated prematurity.
Journal of Clinical Endocrinology & Metabolism 09/2005; 90(9):5279-86. · 6.50 Impact Factor
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ABSTRACT: Obtaining primary human endometrial stromal cells (HESCs) for in vitro studies is limited by the scarcity of adequate human material and the inability to passage these cells in culture for long periods. Immortalization of these cells would greatly facilitate studies; however, the process of immortalization often results in abnormal karyotypes and aberrant functional characteristics. To meet this need, we have introduced telomerase into cultured HESCs to prevent the normal shortening of telomeres observed in adult somatic cells during mitosis. We have now developed and analyzed a newly immortalized HESC line that contains no clonal chromosomal structural or numerical abnormalities. In addition, when compared with the primary unpassaged parent cells, the new cell line displayed similar biochemical endpoints after treatment with ovarian steroids. Classical decidualization response to estradiol plus medroxyprogesterone acetate were seen in both morphologically, and progestin was seen to induce or regulate the expression of IGF binding protein-1, fibronectin, prolactin, tissue factor, plasminogen activator inhibitor-1, and Fas/Fas ligand. In summary, an immortalized HESC line has been developed that is karyotypically, morphologically, and phenotypically similar to the primary parent cells, and it is a powerful and consistent resource for in vitro work.
Endocrinology 06/2004; 145(5):2291-6. · 4.46 Impact Factor
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ABSTRACT: Expression of tissue factor (TF), the primary initiator of hemostasis via thrombin formation, is induced during progesterone (P4)-stimulated decidualization of human endometrial stromal cells (HESCs), and remains elevated in decidualized HESCs of luteal and gestational endometrium. In HESC monolayers, progestins elevate TF mRNA and protein levels and estradiol (E2) plus progestin further enhance TF levels for weeks despite no response to E2 alone. This in vitro model mimics the chronic differential ovarian steroid upregulation of TF levels associated with in vivo decidualization. After incubation of HESCs with E2 plus progestin to elevate TF expression, the antiprogestin RU486 completely reversed this upregulation. Thus, progesterone withdrawal transformed decidualization-associated hemostasis of the luteal phase endometrium to the hemorrhagic milieu of menstruation. Transient transfections with TF promoter constructs containing SP and EGR-1 binding sites before and after inactivation by site-directed mutagenesis revealed that Sp1 mediates basal and progestin-enhanced TF transcriptional activity. Progesterone receptor involvement in TF expression was further confirmed since RU486 was a pure antagonist of progestin-enhanced TF mRNA and protein expression, and progestin-enhanced, but not basal, Sp1-mediated transcriptional activity. Enhanced TF mRNA and protein levels in HESCs require co-incubation with progestin and epidermal growth factor receptor (EGFR) agonist indicating that the EGFR mediates progestin-enhanced TF expression. A peak in the primary angiogenic agent, vascular endothelial growth factor (VEGF) in luteal phase endometrium may be indirectly regulated by P4. Neither E2, nor progestin, nor E2 plus progestin affected VEGF expression in glandular epithelial and stromal cells, whereas thrombin enhanced VEGF mRNA and protein levels in decidualized HESCs, but not in the epithelial cells. Transudation of clotting factors to perivascular decidual cell TF in the luteal phase would generate thrombin, enabling it to act as an autocrine enhancer of VEGF in decidualized HESCs. Abnormal uterine bleeding complicates long-term progestin only contraceptive use. After Norplant administration, endometrial VEGF levels are elevated and TF levels are selectively enhanced in decidualized HESCs at bleeding sites. Over-expressed VEGF causes blood vessels to become leaky, increasing clotting factor access to decidualized HESC-expressed TF to promote feed-forward thrombin and VEGF formation. Since thrombin and VEGF induce angiogenesis via separate endothelial cell receptors, they may synergize to elicit aberrant angiogenesis, and ultimately lead to focal bleeding.
Steroids 12/2003; 68(10-13):849-60. · 2.83 Impact Factor
