Monika Siwetz

Caspases rather than calpains mediate remodelling of the fodrin skeleton during human placental trophoblast fusion

Fusion of cytotrophoblasts with the overlying syncytiotrophoblast is an integral step in differentiation of the human placental villous trophoblast. Multiple factors, such as growth factors, hormones, cytokines, protein kinases, transcription factors and structural membrane proteins, were described to modulate trophoblast fusion. However, the knowledge on remodelling of the membrane-associated cytoskeleton during trophoblast fusion is very limited. This study describes the link between remodelling of spectrin-like α-fodrin and intercellular trophoblast fusion. Experiments with primary trophoblasts isolated from term placentas and the choriocarcinoma cell line BeWo revealed a biphasic strategy of the cells to achieve reorganization of α-fodrin. Syncytialization of trophoblasts was accompanied by down-regulation of α-fodrin mRNA, whereas the full-length α-fodrin protein was cleaved into 120 and 150 kDa fragments. Application of calpeptin and calpain inhibitor III did not affect α-fodrin fragmentation in primary term trophoblasts and forskolin-treated BeWo cells, but decreased secretion of β human chorionic gonadotropin. In contrast, inhibitors of caspases 3, 8 and 9 attenuated generation of the 120 kDa fragment and a general caspase inhibitor completely blocked fragmentation, suggesting an exclusive function of caspases in α-fodrin remodelling. Immunofluorescence double staining of human placenta revealed co-localization of active caspase 8 with α-fodrin positive vesicles in fusing villous cytotrophoblasts. These results suggest that caspase-dependent fragmentation of α-fodrin may be important for reorganization of the sub-membranous cytoskeleton during trophoblast fusion.

Fusion of Villous Trophoblast can be Visualized by Localizing Active Caspase 8

Villous cytotrophoblast differentiation and subsequent fusion with the overlying syncytiotrophoblast depend on multiple factors such as growth factors, cytokines, hormones, protein kinases, transcription factors, structural membrane proteins and proteases. Caspase 8, an aspartate-specific cysteine protease, is mainly known for its role in programmed cell death, but was also demonstrated to be crucial for villous trophoblast differentiation. This study aimed to localize active caspase 8 in the villous trophoblast layer of human first trimester placenta. To this end, immunofluorescence double staining was performed, using a monoclonal rabbit antibody against cleaved caspase 8 in combination with antibodies against cytokeratin 7, chorionic gonadotropin beta subunit (beta hCG), beta-actin, placental protein 13 (PP13), alpha-fodrin and Ki-67. Immunofluorescence revealed cleaved caspase 8 in one out of 422 villous cytotrophoblasts resting on the basement membrane, in one out of 759 perinuclear regions within the syncytiotrophoblast and in few trophoblasts located between these two layers. Double staining of cleaved caspase 8 and Ki-67 antigen revealed that caspase 8 is activated only in cytotrophoblasts which have left the cell cycle. The staining suggests that caspase 8 is activated in villous cytotrophoblasts just prior to fusion of these cells and escorts the nuclei from the mononucleated to the syncytial state.

The art of identification of extravillous trophoblast

Immunohistochemical staining with specific markers for the respective cell type facilitates tracking and identification of cells such as extravillous trophoblast in the uterine wall. Cytokeratin has been recommended as a marker for all kinds of trophoblasts and is commonly used as a marker to identify interstitial as well as endovascular trophoblast. With immunohistochemical double staining of specimens of first trimester placental bed we show that staining with anti-cytokeratin alone is not sufficient to track all routes of trophoblast invasion. Endovascular trophoblasts can be easily mixed up with endoglandular trophoblasts. Thus, additional application of specific markers for extravillous trophoblast such as anti-HLA-G is strongly recommended, ideally in combination with other markers in immunohistochemical or immunofluorescence double staining.

TNF-α alters the inflammatory secretion profile of human first trimester placenta

Implantation and subsequent placental development depend on a well-orchestrated interaction between fetal and maternal tissues, involving a fine balanced synergistic cross-talk of inflammatory and immune-modulating factors. Tumor necrosis factor (TNF)-α has been increasingly recognized as pivotal factor for successful pregnancy, although high maternal TNF-α levels are associated with a number of adverse pregnancy conditions including gestational hypertension and gestational diabetes mellitus. This study describes effects of exogenously applied TNF-α, mimicking increased maternal TNF-α levels, on the secretion profile of inflammation associated factors in human first trimester villous placenta. Conditioned culture media from first trimester villous placental explants were analyzed by inflammation antibody arrays and ELISA after 48 h culture in the presence or absence of TNF-α. Inflammation antibody arrays identified interleukin (IL)-6, IL-8, chemokine (C–C motif) ligand 2 (CCL2), CCL4, and granulocyte-macrophage colony-stimulating factor (GM-CSF) as the most abundantly secreted inflammation-associated factors under basal culture conditions. In the presence of TNF-α, secretion of GM-CSF, CCL5, and IL-10 increased, whereas IL-4 and macrophage CSF levels decreased compared with controls. ELISA analysis verified antibody arrays by showing significantly increased synthesis and release of GM-CSF and CCL5 by placental explants in response to TNF-α. Immunohistochemistry localized GM-CSF in the villous trophoblast compartment, whereas CCL5 was detected in maternal platelets adhering to perivillous fibrin deposits on the villous surface. mRNA-based in situ padlock probe approach localized GM-CSF and CCL5 transcripts in the villous trophoblast layer and the villous stroma. Results from this study suggest that the inflammatory secretion profile of human first trimester placenta shifts towards increased levels of GM-CSF, CCL5, and IL10 in response to elevated maternal TNF-α levels, whereas IL-6 and IL-8 remain unaffected. This shift may represent a protective mechanism by human first trimester villous placenta to sustain trophoblast function and dampen inflammatory processes in the intervillous space.

Placental Fractalkine Is Up-Regulated in Severe Early-Onset Preeclampsia

The pathogenesis of preeclampsia (PE) includes the release of placental factors into the maternal circulation, inducing an inflammatory environment in the mother. One of the factors may be the proinflammatory chemokine fractalkine, which is expressed in the syncytiotrophoblast of human placenta, from where it is released into the maternal circulation by constitutive shedding. We examined whether placental fractalkine is up-regulated in severe early-onset PE and whether the proinflammatory cytokines tumor necrosis factor (TNF)-α and IL-6 are able to increase the expression of fractalkine. Gene expression analysis, enzyme-linked immunosorbent assay, and immunohistochemistry consistently showed increased fractalkine expression in placentas from severe early-onset PE, compared to gestational age-matched controls. Expression of a disintegrin and metalloproteinases (ADAMs) 10 and 17, which convert transmembrane fractalkine into the soluble form, was significantly increased in these cases. Incubation of first-trimester placental explants with TNF-α provoked a significant increase in fractalkine expression and release of the soluble form, whereas IL-6 had no effect. TNF-α-mediated up-regulation of placental fractalkine was reversed in the presence of the aspirin-derivative salicylate, which impaired activation of NF-κB p65 in TNF-α-treated explants. On the basis of data from placental explants, we suggest that increased maternal TNF-α may up-regulate the expression and release of placental fractalkine, which, in turn, may contribute to an exaggerated systemic inflammatory response in PE.

Placental expression of D-chiro-inositol phosphoglycans in preeclampsia.

Abnormalities in glucose metabolism linked to D-chiro-inostol phosphoglycans (IPGs) have been described in human placentas of preeclamptic women. In this study, a semi-quantitative approach to assess the histological assessment of IPGs revealed no significant differences between early and late onset preeclampsia and gestational age matched controls. However, there was a tendency towards higher values in early onset preeclampsia for villous stroma and placental vessels. Moreover, in control cases staining of plasma in placental vessels was present only in one part of vessels of mature intermediate villi while in preeclamptic specimens all placental vessels showed a similar staining. The tendencies of more staining in villous stroma associated with a differential staining of placental vessels only in preeclamptic specimens support a vectoral movement of D-chiro-inositol phosphoglycans from the fetus to the placenta.

Keratins in the human trophoblast.

Besides microfilaments and microtubules, intermediate filaments are major components of the cytoskeleton. In epithelial cells intermediate filaments are formed by heterodimers of specific keratins, whose expression pattern highly depends on the type of epithelium and differentiation degree of the cell. During the process of blastocyst implantation and subsequent development of the human placenta a very specialized epithelium appears at the feto-maternal interface. Arising from the trophectoderm of the blastocyst, the epithelium-like layer surrounding the early embryoblast, different trophoblast subtypes differentiate. They either develop into polar cells fulfilling real epithelial functions, or apolar tumor-like cells invading the maternal uterine wall to adapt the maternal tissue to progressing pregnancy. Thus, the whole trophoblast population, with all its subtypes, can be considered as an epithelial compartment and hence expresses keratin filaments. However, differentiation of trophoblast into different phenotypes may be linked to remodeling of the cytoskeletal composition, depending on spatiotemporal requirements of the respective cells. Here, we focus on the keratin composition of different trophoblast subtypes, how these keratins are used in trophoblast research and what is known about placental keratins in pregnancy pathologies.

Fibulin-5 expression in the human placenta

Fibulin-5 is a secreted extracellular matrix glycoprotein and displays a diverse panel of biological functions, which can be segregated into elastogenic as well as extra-elastogenic functions. While elastogenic functions of fibulin-5 include essential roles in early steps of elastic fibre assembly, extra-elastogenic functions are widespread. Depending on the cell type used, fibulin-5 mediates cell adherence via a subset of integrins, antagonizes angiogenesis and inhibits migration as well as proliferation of endothelial and smooth muscle cells. In this study, we focused on the spatiotemporal expression of fibulin-5 in the human placenta. With progressing gestation, placental fibulin-5 expression increased from first trimester towards term. At term, placental fibulin-5 mRNA expression is lower when compared with other well-vascularized organs such as lung, kidney, heart, uterus and testis. In first trimester, placenta immunohistochemistry localized fibulin-5 in villous cytotrophoblasts and extravillous cytotrophoblasts of the proximal cell column. In term placenta, fibulin-5 was detected in the endothelial basement membrane and adventitia-like regions of vessels in the chorionic plate and stem villi. Cell culture experiments with the villous trophoblast-derived cell line BeWo showed that fibulin-5 expression was downregulated during functional differentiation and intercellular fusion. Moreover, cultivation of BeWo cells under low oxygen conditions impaired intercellular fusion and upregulated fibulin-5 expression. The spatiotemporal shift from the trophoblast compartment in first trimester to the villous vasculature at term suggests a dual role of fibulin-5 in human placental development.

Metalloprotease Dependent Release of Placenta Derived Fractalkine

The chemokine fractalkine is considered as unique since it exists both as membrane-bound adhesion molecule and as shed soluble chemoattractant. Here the hypothesis was tested whether placental fractalkine can be shed and released into the maternal circulation. Immunohistochemical staining of human first trimester and term placenta sections localized fractalkine at the apical microvillous plasma membrane of the syncytiotrophoblast. Gene expression analysis revealed abundant upregulation in placental fractalkine at term, compared to first trimester. Fractalkine expression and release were detected in the trophoblast cell line BeWo, in primary term trophoblasts and placental explants. Incubation of BeWo cells and placental explants with metalloprotease inhibitor Batimastat inhibited the release of soluble fractalkine and at the same time increased the membrane-bound form. These results demonstrate that human placenta is a source for fractalkine, which is expressed in the syncytiotrophoblast and can be released into the maternal circulation by constitutive metalloprotease dependent shedding. Increased expression and release of placental fractalkine may contribute to low grade systemic inflammatory responses in third trimester of normal pregnancy. Aberrant placental metalloprotease activity may not only affect the release of placenta derived fractalkine but may at the same time affect the abundance of the membrane-bound form of the chemokine.

A comparative study of five physiological key parameters between four different human trophoblast-derived cell lines

The human placenta plays a crucial role as the interface between mother and fetus. It represents a unique tissue that undergoes morphological as well as functional changes on the cellular and tissue level throughout pregnancy. To better understand how the placenta works, a variety of techniques has been developed to re-create this complex physiological barrier in vitro. However, due to the low availability of freshly isolated primary cells, choriocarcinoma cell lines remain the usual suspects as in vitro models for placental research. Here, we present a comparative study on the functional aspects of the choriocarcinoma cell lines BeWo, JAR and Jeg-3, as well as the first trimester trophoblast cell line ACH-3P as placental in vitro barrier models for endocrine and transport studies. Functional assays including tight junction immunostaining, sodium fluorescein retardation, trans epithelial resistance, glucose transport, hormone secretion as well as size-dependent polystyrene nanoparticle transport were performed using the four cell types to evaluate key functional parameters of each cell line to act a relevant in vitro placental barrier model.