Yuehong Ma

Thrombin-dependent regulation of matrix metalloproteinase (MMP)-9 levels in human fetal membranes

Objective. Amniochorion matrix metalloproteinase (MMP)-9 levels increase during labor, reaching a maximum in patients with preterm premature rupture of membranes (PPROM). Bleeding is a major risk factor for PPROM. Since such hemorrhage into the tissue factor-enriched decidua induces intense thrombin formation, we determined whether thrombin stimulates MMP levels in amniochorionic membranes. Study design. Fetal membrane (amniochorion) cultures were maintained in media with and without thrombin, lipopolysaccharide (LPS), thrombin receptor agonist peptide (TRAP)-14, and the anti-inflammatory steroid, dexamethasone (DEX). Concentrations of MMP-9, MMP-1, and tissue inhibitor of metalloproteinase (TIMP)-1 in culture media were measured by ELISA and normalized to total cell protein. Results. The presence of thrombin induced MMP-9 levels. TRAP-14, a thrombin receptor agonist, also significantly increased MMP-9 levels, suggesting that thrombin-induced changes in MMP-9 expression were mediated through the thrombin receptor. Conversely, levels of MMP-1 and TIMP-1 were not affected by thrombin treatment, indicative of specificity of its action. The presence of LPS increased the concentration of MMP-9 and MMP-1. In contrast, DEX treatment significantly reduced MMP-9 levels. Conclusion. Our findings clearly demonstrated that thrombin treatment selectively increased the concentration of MMP-9 in culture media of amniochorionic membranes. Our results provide a potential mechanism through which alterations in hemostasis promote PPROM through thrombin-dependent stimulation of MMP-9.

Alterations in Syncytiotrophoblast Cytokine Expression Following Treatment with Lipopolysaccharide

Problem  The placental syncytium is a differentiated cell type on the surface of the villus that has the potential to release cytokines directly to maternal blood. Responsiveness of this cell type to inflammatory compounds remains largely unelucidated.

Glucocorticoid Enhances Transforming Growth Factor-β Effects on Extracellular Matrix Protein Expression in Human Placental Mesenchymal Cells

Abstract Extracellular matrix (ECM) proteins synthesized by human placental mesenchymal cells (PMCs) provide structural support for the villus. Aberrant expression of ECM proteins by PMCs has been associated with intrauterine growth restriction (IUGR). To provide insight into the mechanisms of ECM protein regulation in the stroma of the placental villus, in the current study, we examined the interaction of glucocorticoid (GC) and transforming growth factor-β (TGFβ) in the modulation of ECM proteins in cultures of PMCs isolated from human term placentas. Initial results obtained by ELISA showed that combined treatment with dexamethasone (DEX) and TGFβ enhanced oncofetal fibronectin (FFN) protein levels in serum-free culture medium severalfold in a dose-dependent manner. Northern blotting and real-time polymerase chain reaction (PCR) analyses revealed a similar enhancement in levels of FN mRNA in cells treated with TGFβ and DEX. Real-time PCR results also revealed that DEX and TGFβ enhanced collagen (Col) I and Col IV expression, but did not affect levels of Col III or laminin, indicative of selective stimulation of ECM proteins. Hypoxic treatment moderately enhanced FFN levels in control cells but not in those treated with DEX and TGFβ. In contrast with the results obtained with PMCs, we noted that DEX treatment suppressed FFN levels in untreated and TGFβ-treated cytotrophoblasts, suggesting that GC and TGFβ modulate FFN expression in placenta in a cell-type-specific manner. We conclude that GC and TGFβ are key regulators of ECM protein synthesis in PMCs, suggesting a role in modulating placental architecture in uncomplicated pregnancies and those associated with aberrant ECM protein expression.

Steroid Regulation of Human Placental Integrins: Suppression ofα 2 Integrin Expression in Cytotrophoblasts by Glucocorticoids1

Maintenance of uterine-placental attachment during human pregnancy may depend at least partly on adhesive interactions between cytotrophoblasts and their extracellular matrix (ECM). Such interactions are often mediated by integrins, signal-transducing heterodimeric transmembrane glycoproteins. We previously showed that glucocorticoid (GC) suppressed the expression of collagen and laminin in human placenta; here we show that GC also modulates the expression by human cytotrophoblasts of the integrin subunits alpha2 and beta1, components of a known receptor for these ECM ligands. Cytotrophoblasts were isolated from human term placentas, cultured up to 4 days in the presence of 0-1000 nM dexamethasone (DEX), and assayed for 1) integrin messenger RNA (mRNA) levels by Northern hybridization, 2) integrin subunit synthesis after [35S]methionine labeling, or 3) cell surface integrin levels after 125I labeling by lactoperoxidase. In four independent experiments, 100 nM DEX reduced mRNA levels for integrin alpha2 to 6+/-1% of the control value. This effect was similar between 1-4 days of treatment and was dose dependent between 1-1000 nM DEX. Cortisol treatment (100 nM) inhibited levels of integrin alpha2 mRNA, but 100 nM testosterone, estradiol, and progesterone were less effective, suggesting that this response was specific to GC. In immunoprecipitation studies, treatment of cytotrophoblasts with 100 nM DEX for 2 days reduced the rates of synthesis of the alpha2 integrin subunit as well as its expression on the cell surface to 1-10% of control levels. DEX effects on the beta1 integrin subunit were less dramatic. DEX reduced beta1 mRNA levels to only 69+/-8% of control levels, a smaller reduction compared with effects on alpha2 integrin mRNA. DEX inhibited beta1 protein synthesis and cell surface expression to 60-70% of control levels. In all experiments, DEX had no effect on total protein synthesis. Thus, our results demonstrate that GC treatment specifically and markedly down-regulates expression of alpha2 integrin subunit by human cytotrophoblasts. This finding is consistent with the concept that uterine-placental adherence across gestation may be regulated by coordinate effects on ECM ligands and cellular adhesion receptors.

Differential Effects of Lipopolysaccharide and Thrombin on Interleukin‐8 Expression in Syncytiotrophoblasts and Endothelial Cells: Implications for Fetal Survival

Abstract: Syncytiotrophoblasts (SCTs) are directly bathed by maternal blood and, as such, are in direct contact with proinflammatory stimuli present in the maternal circulation. The extent and nature of cytokine responses induced in SCTs play a central role in the maintenance of pregnancy. Thrombin is a critical mediator of tissue factor‐initiated blood coagulation. Thrombin has been more recently demonstrated to induce cytokine expression and inflammation in several cell types. To dissect the patterns of regulation of cytokine production in the placental villus, we compared the effects of thrombin and lipopolysaccharide (LPS) treatments on cytokine expression in SCTs and endothelial cells. For studies, primary cultures of cytotrophoblasts from human term placentas were differentiated to SCTs. We observed that the presence of thrombin only modestly enhanced interleukin‐8 (IL‐8) levels in SCTs in a manner that was not dose‐dependent. Conversely, SCTs were exquisitely sensitive to LPS, the presence of which induced approximately a 10‐fold increase in IL‐8 levels with an EC50∼ 1 ng/mL. Northern blotting and real‐time PCR results indicated that LPS (but not thrombin) treatment induced a >4‐fold increase in levels of IL‐8 mRNA. The addition of the anti‐inflammatory steroid, dexamethasone, significantly reduced the LPS‐mediated increase in levels of IL‐8 in SCTs. Conversely, in human umbilical vein endothelial cells, thrombin and LPS treatments induced 10‐ and 20‐fold increases in IL‐8 expression, respectively. These results indicate that LPS, but not thrombin, promotes proinflammatory processes in SCTs, with cell‐type specificity. The inability of thrombin in the intervillous space to evoke inflammatory responses in SCTs may constitute an important aspect of fetal survival. Conversely, our results suggest that SCTs do play a key role in infection‐associated changes in placental cytokine expression.

Circulating soluble Fas levels and risk of ovarian cancer

BackgroundDysregulation of apoptosis, specifically overexpression of soluble Fas (sFas), has been proposed to play a role in the development of ovarian cancer. The main objective of the present study was to evaluate serum sFas as a potential biomarker of ovarian cancer risk.MethodsThe association between serum sFas levels and the risk of ovarian cancer was examined in a case-control study nested within three prospective cohorts in New York (USA), Umeå (Sweden), and Milan (Italy). Case subjects were 138 women with primary invasive epithelial ovarian cancer diagnosed between 2 months and 13.2 years after the initial blood donation. Control subjects were 263 women who were free of cancer, and matched the case on cohort, menopausal status, age, and enrollment date. Serum sFas levels were determined using a quantitative sandwich enzyme immunoassay.ResultsSerum sFas levels were similar in women subsequently diagnosed with ovarian cancer (median, 6.5 ng/mL; range, 4.4 – 10.2) and in controls (median, 6.8 ng/mL; range, 4.5 – 10.1). Statistically significant trends of increasing serum sFas with age were observed among cases (r = 0.39, p < 0.0001) and controls (r = 0.42, p < 0.0001). Compared to women in the lowest third, women in the highest third of serum sFas were not at increased risk of ovarian cancer after adjustment for potential confounders (odd ratio (OR), 0.87; 95% confidence interval (CI), 0.42 – 1.82).ConclusionThe results suggest that serum sFas may not be a suitable marker for identification of women at increased risk of ovarian cancer.

Release of oncofetal fibronectin from human placenta.

Oncofetal fibronectin (onfFN) is an extracellular matrix (ECM) protein synthesized by tumours and fetal tissue including placenta. The appearance of onfFN and other cellular FNs in cervico-vaginal secretions and maternal blood is associated with adverse pregnancy outcomes. In the current study, we used dual (maternal and fetal) perfusion of human term placentae and primary cultures of syncytiotrophoblasts (SCTs) to determine whether the human placenta releases onfFN. ELISA and Western blotting revealed that onfFN is preferentially released to the maternal perfusate in a time-dependent manner. Immunodetection with FDC-6, extra domain A (EDA) and cell binding domain-specific antibodies revealed onfFN in maternal perfusate to be a high molecular weight (>450 kDa) protein dimer, similar to that found in amniotic fluid. This suggested that onfFN was released intact from placenta and not cleaved from an ECM. In addition, a similar high molecular weight dimeric onfFN species was noted in conditioned media from cultures of SCTs. Since SCTs directly release proteins to the intervillous space, this suggests that SCTs may be a source of onfFN detected in maternal perfusate. These results indicate that onfFN is released from human placenta and thus levels in maternal sera may provide insight into placental pathophysiology.

Role of the proteasome in the regulation of fetal fibronectin secretion in human placenta.

The goal of the current study was to examine the role of the ubiquitin‐proteasome system (UPS), a pathway of intracellular degradation, in the regulation of fetal fibronectin (FFN) expression in human placenta. Primary cultures of cytotrophoblasts (CTs) and placental mesenchymal cells (PMCs) were isolated from human term placentas and were maintained in serum‐free medium (SFM) in the presence of inhibitors of proteasome‐mediated degradation (e.g., MG132) as well as inhibitors of other proteases. Levels of secreted FFN and interleukin (IL)‐8 in culture media were quantitated by enzyme‐linked immunosorbent assay (ELISA), and cell viability was assessed by trypan blue exclusion. Intracellular levels of FFN and ubiquinated proteins were measured by Western blotting, and levels of fibronectin mRNA were determined following Northern blotting. We found that proteasome inhibitors (MG132, MG262, and PSI) potently suppressed levels of secreted FFN in cultures of CTs, but they not did affect levels of IL‐8. Lysosomal, calpain, and serine protease inhibitors as well as the anti‐inflammatory compound sulfasalazine did not markedly affect levels of secreted FFN in CT cultures. Proteasome inhibitors did not compromise cell viability during the initial 16–18 hours of treatment and did not affect intracellular levels of FFN protein or fibronectin mRNA. The efficacy of suppression of FFN in CT culture media by proteasome inhibitors reflected their effects on intracellular accumulation of ubiquinated proteins. By contrast, the presence of proteasome inhibitors did not alter levels of secreted FFN in cultures of PMCs. We conclude that inhibitors of proteasome‐mediated degradation potently and specifically suppressed extracellular expression of FFN in CTs through a cell type‐specific pathway that did not involve alterations in FFN synthesis. This suggests that accumulation of ubiquinated proteins in the presence of proteasome inhibitors blocks FFN secretion or promotes the extracellular degradation of FFN. This experimental paradigm will be useful for dissecting the role of the UPS in regulating CT function.

Potential Role of Glucocorticoids in the Pathophysiology of Intrauterine Growth Restriction (IUGR)

Although the etiology of intrauterine growth restriction (IUGR) and preeclampsia (PE) remains unclear, most investigators attribute the initial “insult” to poor utero-placental perfusion due to defective trophoblast invasion that ultimately compromises fetal well-being.1, 2, 3 The resultant hypoxia curtails the remodeling of uterine vessels by invasive cytotrophoblasts in the second trimester.1,2 Our results suggest that mediators of fetal stress [i.e., glucocorticoids (GC)] may in fact alter placental gene expression and contribute to the destruction of the placental villous network in pregnancies with IUGR/PE. We will present a molecular model through which GC, induced in response to fetal stress, promotes the placental villous damage observed in pregnancies associated with IUGR/PE. This model incorporates the roles of trophoblast plasminogen activator inhibitor (PAI)-l, mesenchymal extracellular matrix (ECM) proteins, and their regulation by transforming growth factor (TGF)-β. We will employ the term “IUGR/PE” to describe those pregnancies with severely growth-restricted fetuses may also complicated by maternal PE These conditions frequently coexist, and a review of the literature suggests that this placental pathology may be associated with both IUGR and PE. Furthermore, considerable attention has been given to the role of exogenously administered and stress-induced endogenous increases in fetal GC and the development of IUGR. There is mounting evidence that aberrant elevations in GC during fetal life and/or IUGR may result in fetal programming of chronic diseases of adulthood such as diabetes, coronary artery disease, and hypertension.