Homocysteine induces trophoblast cell death with apoptotic features

Hyperhomocysteinemia has been suggested as a possible risk factor in women suffering from habitual abortions, placental abruption or infarcts, preeclampsia, and/or intrauterine growth retardation. However, little is known about the pathogenic mechanisms underlying the action of homocysteine. The present study investigated the in vitro ability of homocysteine to affect trophoblast gonadotropin secretion and to induce cell death. In primary human trophoblast cells, homocysteine treatment (20 micromol/L) resulted in cellular flattening and enlargement, extension of pseudopodia, and cellular vacuolization. Cellular detachment, apoptosis, and necrosis were favored. With in situ nick end labeling, we investigated DNA degradation, and we used M30 CytoDEATH to selectively stain the cytoplasm of apoptotic cells. Cytochrome c release from mitochondria to the cytosol and DNA cleavage in agarose gel have been investigated. Homocysteine, but not cysteine, induced trophoblast apoptosis and significantly reduced human chorionic gonadotropin secretion. These findings suggest that trophoblast cell death might represent a pathogenic mechanism by which homocysteine may cause pregnancy complications related to placental diseases.     

Annexin XI may be involved in Ca2+ - or GTP-gammaS-induced insulin secretion in the pancreatic beta-cell

The aim of this study was to investigate possible involvement of annexin XI in the insulin secretory machinery. In fluorescence immunocytochemistry, annexin XI was found in the cytoplasm of pancreatic endocrine cells and a pancreatic beta-cell line, MIN6, in a granular pattern. MIN6 cells also possessed weak and diffused annexin XI immunoreactivity in the cytoplasm. Immunoelectron microscopy revealed annexin XI in the insulin granules. Insulin secretion from streptolysin-O-permeabilized MIN6 cells was inhibited by anti-annexin XI antibody, when the release was stimulated by either Ca2+ or GTP-gammaS, but not by a protein kinase C-activating phorbol ester. Inhibition of insulin release by anti-annexin XI antibody was reproduced in permeabilized rat islets. These findings suggest that annexin XI may be involved in the regulation of insulin secretion from the pancreatic beta-cells.     

Regulation of progesterone secretion in human syncytiotrophoblast in culture by human chorionic gonadotropin.

In the present investigation we sought to define the specific sites in the pathway of placental progesterone biosynthesis that underlie the action of human chorionic gonadotropin (hCG). When the cells were challenged with dibutryl cAMP (dbcAMP), forskolin or isobutylmethylxanthine, they produced significantly higher amounts of progesterone which in the presence of the hCG antibody was reduced to the level of the control set of cells. Trophoblast cells cultured in serum free medium with 25-hydroxycholesterol (25-OHC) produced increased amounts of progesterone. In the presence of hCG antibody at a concentration which neutralized the secreted hCG, the steroid production was completely blocked, even when the 25-OHC was added to the medium. Also, direct quantitation of the cytochrome P450 SCC enzyme in the absence of hCG indicated a significant decrease. The exogenous addition of low density lipoproteins (LDL) increased the progesterone secretion by the trophoblast cells in culture. Neutralization of hCG by the antibodies, however, drastically reduced the LDL induced progesterone secretion, which was restored by the addition of dbcAMP to the medium. Based on these findings, we suggest a stimulatory effect of hCG on normal trophoblast cells at the level of LDL utilization and cytochrome P450 SCC enzyme. Since dbcAMP could mimic these actions of hCG, the data suggest a possible autocrine/paracrine role of hCG on the trophoblast cells. An additive effect of hCG and cAMP on progesterone secretion observed in our studies, indicate that apart from hCG, adenylate cyclase activity may also be regulated by other factors.     

Immunolocalization and histochemical evidence for the association of two different<Emphasis Type="Italic"> Arabidopsis</Emphasis> annexins with secretion during early seedling growth and development

Annexins are a multigene, multifunctional family of calcium-dependent, membrane-binding proteins found in animal and plant cells. In plants, annexins have been localized in the cytoplasm and at the cell periphery of highly secretory cell types, and in the tip region of polarly growing cells. Consequently, one proposed function for annexins in plant cells is participation in the Golgi-mediated secretion of new wall materials. In Arabidopsis, there are eight different annexin cDNAs, which share between 30% and 81% deduced amino acid sequence identity. We have used two monospecific Arabidopsis anti-annexin antibodies, raised against divergent 31-mer peptides from AnnAt1 and AnnAt2 and a previously characterized pea anti-annexin p35 antibody, for Western blot and immunolocalization studies in Arabidopsis. Western blot analyses of various Arabidopsis protein fractions showed that the two Arabidopsis antibodies are able to specifically recognize annexins in both soluble and membrane fractions. Immunofluorescence results with the three annexin antibodies show staining of secretory cells, especially at the cell periphery in developing sieve tubes, outer root cap cells, and in root hairs, consistent with previous results. In developmentally different stages some staining was also seen near the apical meristem, in some leaf cells, and in phloem-associated cells. Autoradiography following ³H-galactose incorporation was used to more clearly correlate active secretion of wall materials with the localization patterns of a specific individual annexin protein in the same cells at the same developmental stage. The results obtained in this study provide further support for the hypothesis that these two Arabidopsis annexins function in Golgi-mediated secretion during early seedling growth and development.     

Isolation of functional human trophoblast cells and their partial characterization in primary cell culture

Summary Human trophoblast isolation and cell culture procedures were examined to identify variables that enhance secretion of chorionic gonadotropin (HCG) in primary culture. Brief exposure of unminced first-trimester placental specimens to a solution of trypsin-EDTA-DNAse, and isolation of the dispersed cells after Ficoll-hypaque centrifugation yielded primary cultures that were high in HCG secretion and content of epithelial-like cells. The gradual decline in HCG level with time in monolayer culture in these presumptive trophoblast cells was retarded by treatment with theophylline and cyclic adenosine monophosphate. Exposure to methotrexate (MTX) did not increase HCG secretion in normal trophoblast cells, in contrast to a 5-fold stimulation by MTX in the JAR line of choriocarcinoma cells. Clusters of polygonal cells in primary culture progressively lost their capacity to secrete HCG and their epithelial-like morphology. However, they could be maintained as cell strains through approximately 15 passages over a period of 13 to 16 weeks.     

Isolation of function human trophoblast cells and their partial characterization in primary cell culture.

Human trophoblast isolation and cell culture procedures were examined to identify variables that enhance secretion of chorionic gonadotropin (HCG) in primary culture. Brief exposure of unminced first-trimester placental specimens to a solution of trypsin-EDTA-DNAse, and isolation of the dispersed cells after Ficoll-hypaque centrifugation yielded primary cultures that were high in HCG secretion and content of epithelial-like cells. The gradual decline in HCG level with time in monolayer culture in these presumptive trophoblast cells was retarded by treatment with theophylline and cyclic adenosine monophosphate. Exposure to methotrexate (MTX) did not increase HCG secretion in normal trophoblast cells, in contrast to a 5-fold stimulation by MTX in the JAR line of choriocarcinoma cells. Clusters of polygonal cells in primary culture progressively lost their capacity to secrete HCG and their epithelial-like morphology. However, they could be maintained as cell strains through approximately 15 passages over a period of 13 to 16 weeks.     

Anti-idiotype-mediated epitope spreading and diminished phagocytosis by a human monoclonal antibody recognizing late-stage apoptotic cells

Apoptotic cells are considered an important auto-antigenic source in diseases such as systemic lupus erythematosus (SLE). A human monoclonal antibody demonstrating exquisite specificity towards late-stage apoptotic cells was generated from an SLE patient. Polyreactive recognition of ribonucleoproteins Ro52 and Ro60 was observed. The antibody significantly diminished the phagocytosis of apoptotic cells and a concomitant decrease in transforming growth factor-beta (TGF-beta) secretion was observed. Light and heavy chain sequencing revealed the antibody to be in essentially germline configuration. Elicited anti-idiotypic antibodies bound distinct self-antigens and showed augmented reactivity towards apoptotic cells as well. Thus, near-germline encoded antibodies recognizing antigens externalized during the process of apoptosis can mediate a variety of potentially pathogenic effects; decreases in the phagocytic uptake of dying cells would constitute a disease-perpetuating event and stimulation of the idiotypic network could lead to intermolecular epitope spreading, increasing the range of molecular targets..     

Macrophage surface expression of annexins I and II in the phagocytosis of apoptotic lymphocytes

When cells undergo apoptosis, or programmed cell death, they expose phosphatidylserine (PS) on their surface. Macrophages that efficiently phagocytose apoptotic cells also express PS on their surface, although at a lower level. The PS exposed on both cells is required for phagocytosis, because uptake is inhibited by masking PS on either cell with annexin V, a PS-binding protein. The inhibition is not additive, suggesting that the exposed PS molecules on the two cells participate in a common process. We asked whether this dual requirement reflects bridging of the target cell and macrophage by bivalent, PS-binding annexins. Monoclonal antibodies (mAbs) against annexins I or II stained a variety of live phagocytes. Apoptotic Jurkat T lymphocytes and human peripheral T lymphocytes, but not apoptotic thymocytes, were stained by anti-annexin I but not II. Phagocytosis of apoptotic targets was inhibited by mAbs to annexins I or II, or by pretreatment of macrophages with the same mAbs. Pretreatment of apoptotic thymocytes had no effect, whereas pretreating Jurkat cells with anti-annexin I or removing annexin I with EGTA was inhibitory. Annexin bridging is vectorial, because annexin is bound to PS molecules on targets but not on macrophages, suggesting annexins serve as both ligand and receptor in promoting phagocytosis.     

Nucleolar and cytoplasmic localization of annexin V.

The subcellular localization of annexin V in cultured human umbilical vein endothelial cells, epithelial cells and fibroblasts was examined. Indirect immunofluorescence and immunoblotting studies using affinity-purified anti-annexin V antibodies revealed that annexin V is located within the cytoplasm and nucleus of these cells. Further examination and direct binding studies showed that annexin V within the nucleus is associated with the nucleolus. These findings suggest that annexin V may play a role in a nucleolar function, such as ribosome assembly and transport.     

Activated macrophages inhibit human cytotrophoblast invasiveness in vitro

Pre-eclampsia is associated with inadequate cytotrophoblast invasion and remodeling of the uterine spiral arterioles, as well as by an aberrant maternal immune response. This study determined the effect of activated macrophages and one of its products, tumor necrosis factor (TNF)-alpha, on cytotrophoblast invasiveness. Coculture with human lipopolysaccharide-activated macrophages decreased the ability of immortalized HTR-8/ SVneo human trophoblast cells to invade through reconstituted extracellular matrix (P < 0.05). This effect of activated macrophages on trophoblast invasiveness was paralleled by abrogation of a 55-kDa caseinolytic activity corresponding to prourokinase plasminogen activator (pro-uPA) and an increased secretion of plasminogen activator inhibitor 1 (PAI1), as determined by gel zymography and ELISA, respectively. Coculture with nonactivated macrophages did not significantly affect trophoblast invasiveness or pro-uPA and PAI1 secretion. Activated macrophages secreted detectable levels of TNF, and administration of exogenous TNF significantly decreased trophoblast invasiveness (P < 0.05), increased the secretion of PAI1 (P < 0.01), and completely inhibited the pro-uPA-associated caseinolytic activity by binding to the TNF receptor 1. Moreover, addition of up to 10 ng/ml of TNF did not increase the rate of apoptosis in HTR-8/SVneo cells. Finally, the increased secretion of PAI1 by trophoblast cells cocultured with activated macrophages was significantly inhibited when a neutralizing anti-TNF antibody was added to the cocultures. These results suggest that the aberrant presence of activated macrophages around uterine vessels may contribute to inadequate trophoblast invasion and remodeling of the uterine spiral arterioles. Thus, the presence of activated macrophages may be important in the etiology of pre-eclampsia.     

Identification of extra-villous trophoblast cells in human decidua using an apparently unique murine monoclonal antibody to trophoblast

Summary Murine monoclonal antibodies were raised to human first trimester trophoblast cells. Eleven antibodies reacted with first trimester trophoblast, tested by immunoperoxidase staining on frozen sections, but only one had apparent specificity for trophoblast after examining reactivities with a panel of other cells and tissues. This antibody, designated FD0161G, bound selectively to syncytiotrophoblast and non-villous trophoblast in first trimester and term placentae. Villous cytotrophoblast was negative. This was clearly demonstrated on freeze-dried, paraffin embedded tissue sections which have superior architecture to frozen sections. FD0161G reacted with extra-villous trophoblast cells in human decidua which are also delineated by a monoclonal anti-cytokeratin antibody. Unlike the latter, however, FD0161G did not react with decidual glands. Thus FD0161G could be used as a specific probe for extra-villous trophoblast in decidual tissue     

Annexins II and V Inhibit Cell Migration

Cell motility is a crucial component involved in wound healing, development, and tumor metastasis. This study investigated whether extracellular annexins, members of a calcium- and phospholipid-binding family of proteins, play a role in the migration of Lewis lung carcinoma cells. Using assays for wound closure and migration through 8-μm pores, it was found that annexins II and V significantly (>40%) inhibited migration of these highly metastatic cells. Additionally, anti-annexin II antibodies enhanced migration of these same cells in the wound closure assay, while an irrelevant antibody (anti-calmodulin) showed no effect. These effects may be due to annexin–membrane binding and inhibition of phospholipid movement that is necessary for the formation of membrane protrusions.     

Effect of cyclic 3':5'-AMP derivatives prostaglandins and related agents on human chorionic gonadotropin secretion in human malignant trophoblast in culture.

The secretion of human chorionic gonadotropin (hCG) is stimulated by addition of N6, O2'-dibutyryl cyclic 3':5'-AMP (dbcAMP) or theophylline to normal term placenta and human malignant trophoblast cells in vitro. To understand better the specificity of this process, malignant trophoblast cultures were incubated with 3':5'-cyclic AMP (cAMP) derivatives, prostaglandins and other agents for 1 to 3 days, and the secretion of radioimmunoassayable hCG was measured. Whereas dbcAMP was the most potent agent in stimulating secretion of hCG, the N6--and O2'-monobutyryl derivatives of cAMP and phosphodiesterase inhibitors (theophylline, papaverine, 3-isobutyl-1-methylxanthine) also increased the secretion of the hormone. A slight increase in hCG secretion was observed following addition of adenine. By contrast, butyrate, cAMP, cyclic 3':5'-GMP (cGMP), dbcGMP, 5'-AMP, adenosine, L-epinephrine and prostaglandins E1, E2, F1a and F2a were ineffective. Particulate fractions from sonicates of malignant trophoblast cultures contained adenylate cyclase activity which was stimulated more than 10-fold by NaF, but not by either catecholamines or prostaglandins. The relatively specific stimulation of hCG secretion suggested that a regulatory process involving cAMP may have physiological significance in the trophoblast.     

Annexin II-mediated plasmin generation activates TGF-beta3 during epithelial-mesenchymal transformation in the developing avian heart

Epithelial-mesenchymal transformation (EMT), the process by which epithelial cells are converted into motile, invasive mesenchymal cells, is critical to valvulogenesis. Transforming growth factor-beta3 (TGF-beta3), an established mediator of avian atrioventricular (AV) canal EMT, is secreted as a latent complex. In vitro, plasmin-mediated proteolysis has been shown to release active TGF-betas from the latent complex. Annexin II, a co-receptor for tissue plasminogen activator (tPA) and plasminogen, promotes cell-surface generation of the serine protease plasmin. Here, we show that annexin II-mediated plasmin activity regulates release of active TGF-beta3 during chick AV canal EMT. Primary embryonic endocardial-derived cells express annexin II which promotes plasminogen activation in vitro. Incubation of heart explant cultures with either alpha(2)antiplasmin (alpha(2)AP), a major physiological plasmin inhibitor, or anti-annexin II IgG, blocked EMT by approximately 80%, and 50%, respectively. Anti-annexin II IgG-mediated inhibition of EMT was overcome by the addition of recombinant TGF-beta3. Upon treatment with anti-annexin II IgG or alpha(2)AP, conditioned medium from heart explant cultures showed absence of the active fragment of TGF-beta3 by Western blot analysis and a approximately 50% decrease in TGF-beta specific bioactivity. Our results suggest that annexin II-mediated plasmin activity regulates the release of active TGF-beta during cardiac valve development in the avian heart.     

Reversible inhibition of fertility in mice by passive immunization with anticumulus oophorus antibodies

The mucified cumulus oophorus represents an outer enveloping layer around ovulated mammalian oocytes. This coat in its definitive expanded form appears late in the preovulatory development as a result of intensive secretion of intercellular matrix by cumulus cells. We have shown recently that antibodies to the cumulus matrix inhibit human fertilization in vitro. This study was undertaken to assess, in an animal model, the effects of anticumulus oophorus antibodies on fertility by use of different passive immunization protocols. A purified anticumulus immunoglobulin fraction was prepared from hyperimmune rabbit serum and administered at different times before and after mating to mice superovulated with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). A dose-dependent negative effect of this anticumulus antibody preparation on the number of fertilized eggs recovered from the oviducts of treated animals was observed when the antibodies were given before mating. High antibody doses also interfered with oocyte maturation and ovulation if applied on the day of eCG treatment, but no effects on these processes were found when the antibodies were given on the day of hCG treatment. The antifertility effect of anticumulus antibodies was reversible and the antibodies did not affect postfertilization development. These findings make cumulus oophorus antigens serious candidates for the development of a contraceptive vaccine.     

Role of integrin switch and transforming growth factor Beta 3 in hypoxia-induced invasion inhibition of human extravillous trophoblast cells

The physiological hypoxic condition favors the angiogenesis in the placenta. However, it remains unclear how hypoxia regulates the invasion of human extravillous trophoblast cells. In the present study, we first showed that alpha5 integrin expression increased and alpha1 integrin expression decreased in human extravillous trophoblast cells cultured in 1% oxygen as compared with control cells cultured in 8% oxygen. Further data showed that the neutralizing antibody against alpha5 integrin increased the invasion of human extravillous trophoblast cells and the neutralizing antibody against alpha1 integrin inhibited the invasion of human extravillous trophoblast cells. Human extravillous trophoblast cells cultured in 1% oxygen showed reduced invasive capacity, which can be effectively blocked by alpha5 integrin neutralizing antibody. Moreover, human extravillous trophoblast cells exposed to 1% oxygen demonstrated increased expression of transforming growth factor-beta3 (TGFB3), and recombinant human TGFB3 inhibited the invasion of human extravillous trophoblast cells in a dose-dependent manner. The neutralizing antibodies against alpha5 integrin and TGFB3 markedly abrogated hypoxia-induced invasion inhibition in human extravillous trophoblast cells. These data indicate that hypoxia may inhibit the invasion of human extravillous trophoblast cells through inducing the integrin switch from alpha1 integrin to alpha5 integrin and promoting TGFB3 expression.     

Protection by hydroxychloroquine prevents placental injury in obstetric antiphospholipid syndrome

Obstetric antiphospholipid syndrome (OAPS) is mediated by antiphospholipid antibodies (aPLs, and anti‐β2 glycoprotein I antibody is the main pathogenic antibody), and recurrent abortion, preeclampsia, foetal growth restriction and other placental diseases are the main clinical characteristics of placental pathological pregnancy. It is a disease that seriously threatens the health of pregnant women. Hydroxychloroquine (HCQ) was originally used as an anti‐malaria drug and has now shown benefit in refractory OAPS where conventional treatment has failed, with the expectation of providing protective clinical benefits for both the mother and foetus. However, its efficacy and mechanism of action are still unclear. After clinical data were collected to determine the therapeutic effect, human trophoblast cells in early pregnancy were prepared and treated with aPL. After the addition of HCQ, the proliferation, invasion, migration and tubule formation of the trophoblast cells were observed so that the therapeutic mechanism of HCQ on trophoblast cells could be determined. By establishing an obstetric APS mouse model similar to the clinical situation, we were able to detect the therapeutic effect of HCQ on pathological pregnancy. The normal function of trophoblast cells is affected by aPL. Antibodies reduce the ability of trophoblast cells to invade and migrate and can impair tubule formation, which are closely related to placental insufficiency. HCQ can partially reverse these side effects. In the OAPS mouse model, we found that HCQ prevented foetal death and reduced the incidence of pathological pregnancy. Therefore, HCQ can improve pregnancy outcomes and reverse the aPL inhibition of trophoblast disease. In OAPS, the use of HCQ needs to be seriously considered.     

K+ channel inhibition modulates the biochemical and morphological differentiation of human placental cytotrophoblast cells in vitro

Maintaining placental syncytiotrophoblast, a specialized multinucleated transport epithelium, is essential for normal human pregnancy. Syncytiotrophoblast continuously renews through differentiation and fusion of cytotrophoblast cells, under paracrine control by syncytiotrophoblast production of human chorionic gonadotropin (hCG). We hypothesized that K(+) channels participate in trophoblast syncytialization and hCG secretion in vitro. Two models of normal-term placenta were used: 1) isolated cytotrophoblast cells and 2) villous tissue in explant culture. Cells and explants were treated with K(+) channel modulators from 18 h, and day 3, onward, respectively. Culture medium was analyzed for hCG, to assess secretion, as well as for lactate dehydrogenase (LDH), to indicate cell/tissue integrity. hCG was also measured in cytotrophoblast cell lysates, indicating cellular production. Syncytialization of cytotrophoblast cells was assessed by immunofluorescent staining of desmosomes and nuclei. Over 18-66 h, mononucleate cells fused to form multinucleated syncytia, accompanied by a 28-fold rise in hCG secretion. 1 mM Ba(2+) stimulated cytotrophoblast cell hCG secretion at 66 h compared with control, whereas 5 mM tetraethylammonium (TEA) inhibited hCG secretion by >90%. 0.1-1 mM 4-aminopyridine (4-AP) reduced cytotrophoblast cell hCG secretion and elevated cellular hCG; without altering cellular integrity or syncytialization. In villous explants, hCG secretion was not altered by 1 mM Ba(2+) but inhibited by 5 mM 4-AP and 5/10 mM TEA, without affecting LDH release. Anandamide, pinacidil, and cromakalim were without effect in either model. In conclusion, 4-AP- and TEA-sensitive K(+) channels (e.g., voltage-gated and Ca(2+)-activated) regulate trophoblast hCG secretion in culture. If these K(+) channels participate in hCG secretion in situ, they may regulate trophoblast turnover in health and disease.     

Induction of p38 mitogen-activated protein kinase-mediated apoptosis is involved in outgrowth of trophoblast cells on endometrial epithelial cells in a model of human trophoblast-endometrial interactions

During embryo implantation in species with hemochorial placentation, such as the mouse and human, trophoblast cells of the attached blastocyst penetrate the luminal epithelium of the endometrium before invasion into the endometrial stroma. Signs of apoptosis were demonstrated in luminal endometrial epithelial cells (EEC) adjacent to the trophoblast cells; however, the signaling mechanisms leading to apoptosis in EEC remain unclear. Because mitogen-activated protein kinases (MAPK) were shown to mediate apoptosis in several model systems and found to be activated in the uterus during decidualization, the possible involvement of MAPK during trophoblast-EEC interactions was studied. By coculturing BeWo human trophoblast spheroids with RL95-2 human EEC monolayers to mimic the blastocyst-endometrial interaction, we found that most spheroids rapidly attached to EEC monolayers and then progressively expanded, with marked dislodgment of EEC adjacent to the spreading trophoblast cells. Immunoblotting analysis showed that both p38 MAPK and extracellular signal-regulated kinase (ERK) were activated in EEC after coculture. However, only SB203580 (a p38 MAPK inhibitor), but not PD98059 (an ERK inhibitor), inhibited trophoblast outgrowth on EEC monolayers through the suppression of p38 MAPK activation in EEC. Furthermore, trophoblast expansion caused prominent EEC apoptosis at the spheroid-EEC interface, as detected by annexin V labeling and valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (which binds activated caspases) staining, and SB203580 significantly decreased the percentage of apoptotic cells. Our results, based on a model of human trophoblast-EEC interactions, establish that trophoblast cells cause activation of p38 MAPK in EEC and, consequently, induce apoptosis and displacement of EEC, a process that may facilitate implantation.