Caspase 8 and human villous cytotrophoblast differentiation.

The fusion of villous cytotrophoblast into the placental syncytium is the quintessential process in maintenance of a healthy pregnancy. Efficient fusion requires a phosphatidylserine (PS)-rich cytotrophoblast surface, expression of fusion proteins, cessation of cell cycling, and rearrangement of cytoskeleton to accommodate membrane joining. Significant debate surrounds the potential role of apoptosis-related proteins, particularly caspase 8. The hypothesis that caspase 8 proteolytic activity is required for villous cytotrophoblast syncytialization rests on a foundation of three specific claims; cytotrophoblast PS efflux is an indication of early apoptosis, caspase 8 activation precedes intercellular fusion, and inhibition of caspase 8 proteolytic activity diminishes syncytialization. Our analysis of these claims reveals weaknesses that justify a reevaluation of the role of caspase 8 in villous cytotrophoblast fusion. In models of physiologic intercellular fusion, including villous cytotrophoblast, PS efflux is unrelated to apoptosis and is controlled by ATP-dependent transporters. Only a small amount of prefusion activation of caspase 8 occurs in mononuclear cytotrophoblast, and the significance remains controversial. Specific caspase 8 inhibitions with specific peptide inhibitors or antisense oligonucleotides or silencing with siRNA substantiate potential differentiation-related roles, unrelated to initiation of intercellular fusion, for both procaspase 8 and activated caspase 8. From this analysis a new and testable model of villous cytotrophoblast differentiation and fusion is presented.

In vitro evidence that hyperglycemia stimulates tumor necrosis factor-alpha release in obese women with polycystic ovary syndrome.

Women with polycystic ovary syndrome (PCOS) are often insulin resistant and have chronic low-level inflammation. The purpose of this study was to determine the effects of hyperglycemia in vitro on tumor necrosis factor (TNF)-alpha release from mononuclear cells (MNC) in PCOS. Twelve reproductive-age women with PCOS (six lean, six obese) and 12 age-matched controls (six lean, six obese) were studied. Insulin sensitivity (IS(HOMA)) was estimated from fasting levels of glucose and insulin and percent truncal fat was determined by dual energy absorptiometry (DEXA). TNFalpha release was measured from MNC cultured under euglycemic and hyperglycemic conditions. IS(HOMA) was higher in obese women with PCOS than in lean women with PCOS (student's t-test; 73.7 +/- 14.8 vs 43.1 +/- 8.6, P < 0.05), but similar to that of obese controls. IS(HOMA) was positively correlated with percent truncal fat (r=0.57, P < 0.04). Obese women with PCOS exhibited an increase in the percent change in TNFalpha release from MNC in response to hyperglycemia compared with obese controls (10 mM, 649 +/- 208% vs 133 +/- 30%, P < 0.003; 15 mM, 799 +/- 347% vs 183 +/- 59%, P < 0.04). The TNFalpha response directly correlated with percent truncal fat (r=0.45, P < 0.03) and IS(HOMA) (r=0.40, P < 0.05) for the combined groups, and with plasma testosterone (r=0.60, P < 0.05) for women with PCOS. MNC of obese women with PCOS exhibit an increased TNFalpha response to in vitro physiologic hyperglycemia. MNC-derived TNFalpha release may contribute to insulin resistance and hyperandrogenism, particularly when the combination of PCOS and increased adiposity is present.

The role of human endogenous retroviruses in trophoblast differentiation and placental development.

A major portion of the human genome appears to be of retroviral origin. These endogenous retroviral elements are expressed in a variety of normal tissues and during disease states, such as autoimmune and malignant conditions. Recently, potential roles have been described for endogenous retroviral envelope proteins in normal differentiation of human villous cytotrophoblast into syncytiotrophoblast. This article provides a brief critical review of the current state of knowledge concerning the expression of the env regions of three endogenous retroviral elements: ERV-3, HERV-W, and HERV-FRD. A testable model of villous cytotrophoblast differentiation is constructed, in which a complementary expression of endogenous retroviral envelope proteins initiates hCG production, decreased cell proliferation, and intercellular fusion.

Phosphatidylserine efflux and intercellular fusion in a BeWo model of human villous cytotrophoblast.

Phosphatidylserine (PS) efflux characterizes cytotrophoblast apoptosis and differentiation. To evaluate whether PS externalization and intercellular fusion were secondary to apoptosis, BeWo cells were induced to differentiate by forskolin or undergo apoptosis by staurosporine. PS externalization was measured by FITC-annexin V binding, and intercellular fusion was quantified by counting nuclei in syncytial cells. During forskolin treatment, vanadate decreased PS efflux by 78.0 per cent from 68.0 [5.3] (mean [SD]) to 15.0 [8.8] Lum (x10(3)) (P<0.001), whereas Z-VAD-fmk had no effect (66.5 [7.3]). Vanadate decreased intercellular fusion from 78.1 per cent [4.1] fusion in uninhibited cultures to 23.4 per cent [2.5], compared with 10.0 per cent [1.7] in media alone. Z-VAD-fmk did not affect fusion (80.4 per cent [6.8]). Staurosporine induced PS efflux was not affected by vanadate (69.6 [5.5] Lum x10(3)), but was inhibited 87.8 per cent by Z-VAD-fmk; from 71.5 [6.2] to 8.7 [3.6] Lum (x10(3)) (P<0.001). Apoptosis was measured by the TUNEL and COMET assays, lamin B fragmentation, activation of procaspase 3, mitochondrial membrane potential, and release of mitochondrial cytochrome c and apoptosis inducing factor. There was no indication of apoptosis associated with differentiation. Thus, PS efflux and intercellular fusion occurred through a vanadate-sensitive mechanism that was independent of apoptosis.

Preparation and functional characterization of villous cytotrophoblasts free of syncytial fragments.

Recent studies suggest that purified villous cytotrophoblasts are largely contaminated by mononucleated syncytial fragments and therefore unsuitable for studies of trophoblast differentiation. We assessed highly purified (>99.99 per cent) populations of villous trophoblasts for fragment contamination using the syncytial markers placental alkaline phosphatase (PLAP, by immunohistochemistry) and exteriorized phosphatidyl serine (ePS, by flow cytometric analysis). The preparations contained from 4-46 per cent syncytial fragments. However, we find that PLAP negative cells preferentially adhere to tissue culture surfaces and that all preparations were <2 per cent PLAP positive after routine plating and washing procedures. A second purification procedure eliminated dead (propidium iodide permeable) cells and separated viable syncytial fragments (ePS-positive) from viable cytotrophoblasts (ePS-negative) by two colour fluorescence activated cell sorting (FACS). Viable ePS-positive cells were ultrastructurally apoptotic, adhered poorly in culture and those that adhered rapidly underwent apoptosis. Viable ePS-negative cells contained large heterochromic nuclei and cytoplasmic structures, adhered strongly in culture and remained viable. The latter population (putative true villous CT) differentiated into syncytialized cells when cultured with EGF. We conclude that villous CT can be routinely purified, are viable in culture and can undergo syncytial fusion without extensive preformed syncytium.

Ultrastructural localization of monoclonal antiphospholipid antibody binding to rat brain.

Antiphospholipid antibodies (aPL), in the presence or absence of systemic lupus erythematosus, are associated with a number of neurologic complications. However, the role aPL play in pathology is unclear. A thrombotic etiology seems likely for many associated disorders, but not for others. Here we describe aPL-reactive sites in the central nervous system (CNS). Previously, using light microscopy, we showed direct binding of two monoclonal phosphatidylserine-reactive antibodies (aPS) to ependyma and myelin of fixed cat brain. In this study we determined the ultrastructural localization of their binding sites in rat CNS using immunogold electron microscopy techniques. Both monoclonal antibodies reacted strongly with myelin, preferentially with the major dense line formed by the cytoplasmic apposition of the oligodendrocyte plasma membrane. Both monoclonal antibodies also reacted with an antigen that appears associated with the axoneme in cilia of ependymal and choroid plexus epithelium. One monoclonal aPS also showed some reactivity with brain vascular endothelium and reacted slightly with mitochondria, while the other aPS did not react with these structures. While the etiology of aPL-associated neurologic disorders remains unclear, our data suggest possible target sites within the CNS with which aPL can react.

The cellular mechanism by which the human endogenous retrovirus ERV-3 env gene affects proliferation and differentiation in a human placental trophoblast model, BeWo.

The env region of the human endogenous retrovirus ERV-3 is expressed during differentiation of trophoblast and the choriocarcinoma BeWo. Stable transfectants with ERV-3 env exhibit most aspects of trophoblast differentiation, including inhibition of cell proliferation, changes in cell morphology, and increased production of beta-hCG mRNA. In this study, the cellular mechanism of induction of BeWo cell differentiation by ERV-3 env was investigated. In BeWo cells stably transfected with ERV-3 env, the production of beta-hCG mRNA and hCG protein was increased. Intracellular cAMP level was markedly increased over that of vector transfected cells. The effect on beta-hCG protein production was inhibited by H89, a protein kinase A (PKA) inhibitor, while protein kinase C (PKC) and protein tyrosine kinase (PTK) inhibitors had no effect. The expression of a major cell cycle promoter, cyclin B, was markedly reduced while expression of p21, a negative regulator of the cell cycle, was up-regulated. Inhibition of ERV-3 env induced hCG production with H89 had no significant effect on cell growth when compared with cells transfected with vector alone.

Identification of a stress-induced protein during human trophoblast differentiation by differential display analysis.

Differentiation of human placental trophoblast is characterized by a process during which mononuclear villous cytotrophoblasts fuse to form a multinucleate syncytium. This event is associated with dramatic changes in gene expression. In the present study, we have applied a sensitive approach-differential display analysis-to evaluate changes in gene expression during in vitro forskolin-induced differentiation of a model of human trophoblast, the choriocarcinoma BeWo. We identified seven genes that were up-regulated; their expression and function have not previously been reported in trophoblast. Four up-regulated genes were novel upon comparison of their sequences to the GenBank database. The other three genes encode human cytochrome p450 IIC, inosine monophosphate dehydrogenase type II, and reducing agent and tunicamycin-responsive protein (RTP). Northern blot analysis revealed that RTP mRNA expression was induced to 3-fold in BeWo after 24-h incubation with forskolin and increased up to 11-fold by 72 h of forskolin treatment. The expression pattern of RTP was further investigated by in situ hybridization on second trimester and term placenta tissues. RTP mRNA was predominantly expressed in syncytiotrophoblasts in both second trimester and term placentae. The expression of RTP gene in BeWo cells was protein kinase C dependent. This is the first description of RTP gene expression in placenta and the first study elucidating the signaling pathway involved in the regulation of RTP gene expression. These results suggest that RTP may play a role in trophoblast cell proliferation and differentiation.

Antiphospholipid antibodies and reproduction: the antiphospholipid antibody syndrome.

In women who have a diagnosis of APS (both clinical and laboratory criteria) the chance for successful pregnancy is reduced. In these cases, treatment appears to be a clear option, particularly in the case of prior thromboembolic events. The current preference of treatment for women with RPL and aPL antibodies is subcutaneous heparin and aspirin. This treatment should begin with a positive pregnancy test and continue postpartum. It is unclear, at this time, what treatment, if any, is required for women who do not meet all the criteria for diagnosis of APS, but who are known to have aPL antibodies. In some cases, these women were tested because of a prior false-positive test for syphilis, with subsequent identification of aPL antibodies. More recently, women undergoing IVF were tested and found to have an increased incidence of aPL antibodies. It was suggested that aPL antibodies are associated with infertility and failure to implant. However, a summary of published reports indicate that positive aPL antibodies in patients undergoing IVF do not influence ongoing pregnancy rates. This subject, however, remains an area of active investigation because aPL antibodies were shown to interact with the syncytiotrophoblast and cytotrophoblast layers and could, theoretically, after implantation.

Characterization of antigens expressed in normal baboon trophoblast and cross-reactive with HIV/SIV antibodies.

Electron microscopic studies have revealed the presence of endogenous retroviral (ERV) particles in normal primate placental tissues. These particles have ultrastructural similarities to type C retroviral particles and are mainly associated with the trophoblast. In normal human placental tissues, they have antigenic similarity with exogenous retroviruses, such as the human immunodeficiency virus (HIV), and may have a role to play in the regulation of cellular gene expression, syncytiotrophoblast formation or pregnancy-related immunosuppression. In this study, a panel of antibodies (polyclonal and monoclonal antibodies) against viral proteins (anti-HIV and anti-SIV) and endogenous retroviral (ERV) proteins were assessed by immunohistochemistry and immunoblotting, for their cross-reactivity with ERV particles isolated from normal baboon placental tissues. The antibodies (anti-HERV-K RT, anti-ERV3 env, anti-HIV-1 p17, anti-HIV-2 gp120) reacted positively with the syncytiotrophoblast and each antibody recognized one or two proteins of molecular weights (MW) 38, 58 or 64 kDa present in the baboon placental villous tissues and SIV-infected molt-4 Cl8 cells, but not in uninfected cells. The results of this study confirm the specific expression of retroviral cross-reactive antigens in normal baboon placental tissues and suggest placental cellular proteins may have antigenic similarity with those recognized by anti-HIV/SIV antibodies. The role of these retroviral-related proteins expressed at the maternal-fetal interface remain unclear.

Expression of endogenous retrovirus ERV-3 induces differentiation in BeWo, a choriocarcinoma model of human placental trophoblast.

Differentiation-related expression of endogenous retrovirus ERV-3 env in the normal human placental syncytiotrophoblast suggests a role in placental development. The choriocarcinoma cell line BeWo, a model of trophoblast differentiation, is maintained in an undifferentiated state and undergoes differentiation upon the addition of forskolin. The expression of ERV-3 env mRNA increased after 48 h forskolin treatment, concurrently with increased intercellular fusion and production of human chorionic gonadotropin (beta-hCG) mRNA, a hormonal differentiation marker for trophoblast. Over expression of ERV-3 env induced differentiation of BeWo characterized by decreased cell growth, differentiation-related morphologic changes, and induction of beta-hCG mRNA. These results support the first known role for the expression of an endogenous retrovirus in trophoblast differentiation.

Molecular characterisation of a putative endogenous retrovirus cDNA isolated from human placental tissue.

The human genome comprises of abundant DNA sequences related to endogenous retroviruses (ERV) and a variety of solitary long terminal repeats (LTRs). Substantial numbers of intact retroviral particles have been detected by electron microscopy in normal human placental villous tissue particularly in syncytiotrophoblast. Understanding the molecular structure, organisation and distribution of these ERV sequences may lead to elucidation of their possible dual function at the foetal-maternal interface; proliferation and differentiation of cytotrophoblast and induction of local pregnancy-associated immune suppression thus allowing survival of the foetal allograft. In this study, antibody probes were used to screen a human placental expression library and cDNA clones isolated were characterized by polymerase chain reaction, Southern blot hybridisation, DNA cloning and partial nucleotide sequencing. A specific 1.7kb-cDNA clone was isolated from a human placental expression library. Further characterisation showed this clone represents a single copy gene, approximately 9-10kb and did not hybridise to the env region of ERV3 human endogenous retrovirus. The 1.7kb-cDNA clone may represent a provirus co-expressed with cellular sequences.

Molecular Characterisation of a Putative Endogenous Retrovirus cDNA Isolated from Human Placental Tissue

The human genome comprises of abundant DNA sequences related to endogenous retroviruses (ERV) and a variety of solitary long terminal repeats (LTRs). Substantial numbers of intact retroviral particles have been detected by electron microscopy in normal human placental villous tissue particularly in syncytiotrophoblast. Understanding the molecular structure; organisation and distribution of these ERV sequences may lead to elucidation of their possible dual function at the foetal-maternal interface; proliferation and differentiation of cytotrophoblast and induction of local pregnancy-associated immune suppression thus allowing survival of the foetal allograft. In this study; antibody probes were used to screen a human placental expression library and cDNA clones isolated were characterized by polymerase chain reaction; Southern blot hybridisation; DNA cloning and partial nucleotide sequencing. A specific 1.7kb-cDNA clone was isolated from a human placental expression library. Further characterisation showed this clone represents a single copy gene; approximately 9-10kb and did not hybridise to the env region of ERV3 human endogenous retrovirus. The 1.7kb-cDNA clone may represent a provirus co-expressed with cellular sequences

The role of placental trophoblast in the pathophysiology of the antiphospholipid antibody syndrome.

PROBLEM: The antiphospholipid (aPL) antibody syndrome is characterized by severe pregnancy complications, the cause of which remains unknown. We hypothesized that the placental trophoblast is a target for aPLs. METHOD OF STUDY: The effects of monoclonal aPLs on trophoblast function, including the invasion of JAR into matrigel-coated filters and the effects of annexin V expression on BeWo, were investigated using choriocarcinoma models. RESULTS: aPLs against phosphatidylserine (PS) significantly (P < 0.001) decreased the migration of JAR across the membrane. In the annexin V studies, undifferentiated BeWo did not express surface annexin V. After differentiation, BeWo expressed surface annexin V, which was removed in the presence of aPLs, resulting in increased binding of prothrombin. CONCLUSIONS: PS is expressed on the trophoblast surface during differentiation and invasion of extracellular matrix. Our data suggest that aPLs against PS can directly affect trophoblast function by limiting the depth of decidual invasion and by concurrently creating a procoagulant surface on trophoblast exposed to the maternal circulation.

Immunohistochemical localization of retroviral-related antigens expressed in normal baboon placental villous tissue.

Endogenous retroviral particles (ERVs) have been detected in the genome of all eukaryotes. They are generally non-pathogenic except in mice where they have been found to induce tumors and immunological disorders. The ERVs have morphological features consistent with type-C retroviral particles and are commonly expressed in normal placental villous tissues. ERVs may have a role in the regulation of placental gene expression, syncytiotrophoblast formation, or pregnancy-related immunosuppression. In this study, well-characterized antibodies (monoclonal and polyclonal antibodies) raised against retroviral proteins (anti-HIV and anti-SIV) and endogenous retroviral (ERV) particles were assessed for their cross-reactivity (by using immunohistochemistry) with normal baboon placental and other adult tissues. The monoclonal antibodies to exogenous retroviral proteins (anti-HIV-2 gp120, anti-HIV-1 gp41, anti-SIVmac p27, anti-HIV-1 RT, and anti-HIV-2 core protein) showed specific immunohistochemical reactivity with the syncytiotrophoblast. Antibodies to endogenous retroviral gene products (anti-ERV3 env, anti-HERV-K RT, and anti-HERV-K env) also reacted in a similar manner and did not cross-react with other adult tissues. These studies have shown that retroviral-cross-reactive proteins are expressed in baboon placental syncytiotrophoblast and may have a role to play at the feto-maternal interface.

Antiphosphatidylserine antibody removes annexin-V and facilitates the binding of prothrombin at the surface of a choriocarcinoma model of trophoblast differentiation.

OBJECTIVE: Trophoblast differentiation is associated with externalization of phosphatidylserine from the inner to the outer surface of the plasma membrane. In this study we tested the hypothesis that concurrent externalization and binding of annexin-V blocks the phosphatidylserine-rich surface from acting as a site for activation of coagulation and that antiphospholipid antibodies lead to a procoagulant state by preventing annexin-V binding. STUDY DESIGN: A choriocarcinoma model of trophoblast differentiation, forskolin-activated BeWo cells and immunoperoxidase techniques were used to determine surface and cytoplasmic localization of annexin-V related to differentiation. Monoclonal immunoglobulin M antibodies against phosphatidylserine- and cardiolipin-dependent antigens were used to determine the effects of antiphospholipid antibodies on annexin-V localization and on the binding of prothrombin to the BeWo surface. RESULTS: During differentiation BeWo cells externalized phosphatidylserine and increased the expression of surface annexin-V. Monoclonal antibody against phosphatidylserine removed annexin-V from the BeWo surface and increased binding of prothrombin. CONCLUSION: Antiphosphatidylserine antibody induces sites for prothrombin binding on the surface of a BeWo model of trophoblast, most likely by removing annexin-V. This mechanism could explain the frequent observation of increased thrombosis at the maternal-fetal interface in miscarriages associated with antiphospholipid antibodies.