Bonnie Winkler-Lowen

Epidermal growth factor inhibits cytokine-induced apoptosis of primary human trophoblasts

In the placenta, as in other organs, the development and maintenance of the differentiated phenotype depend on a balance between cell proliferation, maturation, and death. We are interested in the mechanisms that regulate the survival and differentiation of placental trophoblasts and have recently demonstrated that the inflammatory cytokines tumor necrosis factor alpha (TNFα) and gamma interferon (IFNγ) act in concert to induce apoptotic cell death in normal cytotrophoblasts in culture. In this report we show that exposure to epidermal growth factor (EGF), a 6,700 dalton polypeptide that is abundantly expressed in maternal and fetal tissues, blocks the in vitro TNF/IFN‐induced cytotoxicity of human cytotrophoblasts and syncytiotrophoblasts from normal term placentas. This antagonistic effect is dose‐related (10−10 M EGF, half‐maximal) and proceeds via the interruption of an early step in the cytokine‐induced apoptotic response. These observations suggest a novel role for EGF in normal placental development and indicate that the interplay between EGF, TNFα, and IFNγ may determine the rate of trophoblast growth and renewal during gestation.

Infection of Placental Trophoblasts by Toxoplasma gondii

How the intracellular parasite Toxoplasma gondii causes placental inflammation and infects the fetus is unknown. By use of a culture model of primary human trophoblasts, we examined the consequences of infection by a virulent strain of T. gondii. Infection fractions (parasitophorous vacuoles per trophoblast nuclei) < or =0.9 were observed 1 day after challenge at an inoculum ratio of T. gondii to nuclei of 10. The culture content of infectious T. gondii increased 45-fold in 48 h. Two days after infection, almost 30% of trophoblast nuclei became apoptotic, and 30%-35% of nuclei were lost. Almost 90% of apoptotic nuclei were not adjacent to a parasitophorous vacuole, suggesting infection protected against apoptosis. However, there was no T. gondii-dependent accumulation of putative cytotoxic factors, such as tumor necrosis factor-alpha, that could mediate paracrine killing. Both mature and immature trophoblasts can be productively infected, and uninfected, but not infected, cells undergo apoptosis.

Polarized Release of Matrix Metalloproteinase-2 and -9 from Cultured Human Placental Syncytiotrophoblasts1

Abstract The large increase in placental surface area and fetal villous vascular development in the third trimester of pregnancy requires degradation and reformation of the placental basal lamina. Degradation is carried out by matrix metalloproteinases (MMPs) secreted by adjacent cells. Although the gelatinases, MMP-2 and MMP-9, which are released by extravillous cytotrophoblasts (CTs) are believed to play crucial roles in early placental expansion, neither has been reported in third trimester villous trophoblasts nor has appropriate (basolateral) release of any MMP by the highly polarized syncytiotrophoblast (ST) been demonstrated. We demonstrated villous trophoblast expression of both MMP-2 and MMP-9 by in situ immunohistochemistry and by Western blot analysis and zymography of lysates and culture supernatants of highly purified villous CTs. We also found that epidermal growth factor (EGF)-stimulated CT differentiation into ST and stimulation by the phorbol diester, PMA, both increase MMP-9 secretion. The direction of MMP release was determined with confluent cultures of ST on porous membranes. We found that >90% of MMP-2 and MMP-9 were released from the basolateral surface. We conclude that villous STs express and release gelatinases from their basolateral surfaces in a regulated manner and suggest that such polarized release may be important to villous tissue remodeling.

Differential proteomic analysis of highly purified placental cytotrophoblasts in pre-eclampsia demonstrates a state of increased oxidative stress and reduced cytotrophoblast antioxidant defense.

Proteomics were performed using highly (99.99%) purified cytotrophoblasts from six normal and six pre‐eclamptic placentas. Eleven proteins were found which decreased in pre‐eclampsia (actin, glutathione S‐transferase, peroxiredoxin 6, aldose reductase, heat shock protein 60 (Hsp60), two molecular forms of heat shock protein 70 (Hsp70) β‐tubulin, subunit proteasome, ezrin, protein disulfide isomerase, and phosphoglycerate mutase 1). Only one protein, α‐2‐HS‐glycoprotein (fetuin), was found to increase its expression. Western blots of actin, Hsp70, ezrin, and glutatione S‐transferase confirmed decrease in protein expression. Many of the proteins that decreased are consistent with a state of oxidative stress in the pre‐eclamptic placenta and a decreased cytotrophoblast defense against and response to oxidative stress.

Adrenomedullin Is Decreased in Preeclampsia Because of Failed Response to Epidermal Growth Factor and Impaired Syncytialization

Abstract—To explore the mechanisms of adrenomedullin (ADM) regulation in normal and preeclamptic (PE) states, we determined placental production of ADM and ADM regulation by cytokines. Isolated, purified cytotrophoblast cultures from normal (n=8) and PE (n=10) placentas were cultured for 3 days in the absence or presence of 10 ng/mL epidermal growth factor (EGF), 1 ng/mL transforming growth factor (TGF)-&bgr;1, 10 ng/mL tumor necrosis factor (TNF)-&agr;, or 100 U/mL interferon (IFN)-&ggr;. Cells were also cultured for 3 days in 10% fetal bovine serum for determination of syncytial formation by desmoplakin staining. Pieces of normal and PE placentas were snap-frozen for ADM mRNA measurement. Results showed that basal ADM production into culture medium by radioimmunoassay was significantly lower in PE placental cells. EGF significantly stimulated ADM production in normal trophoblasts but did not in PE placentas. None of the factors TNF-&agr;, TGF-&bgr;1, or IFN-&ggr; altered ADM secretion in either normal or PE placentas. ADM expression by Northern blot analysis demonstrated a 34.3±8.3% reduction in mRNA expression in PE placentas. Syncytialization, as assessed by desmoplakin-outlined syncytial units, was decreased in PE placentas (day 3: normal, 16.7±1.3%; PE, 5.5±2.0%; P <0.01, ANOVA). However, there was a normal increment in syncytialization in response to EGF in normal and PE trophoblast preparations (EGF day 3: normal, 43.8±5.6%; PE, 46.1±12.3%). We conclude that spontaneous placental syncytialization is impaired in PE and that ADM production is markedly reduced in PE, possibly owing to an impaired EGF response. These abnormalities indicate poor placental production of ADM as the likely cause of a failed compensatory increase in maternal serum ADM levels in PE.

Analysis of the synergistic stimulation of mouse macrophage proliferation by macrophage colony-stimulating factor (CSF-1) and tumor necrosis factor alpha (TNF-alpha).

Abstract: Tumor necrosis factor a (TNF‐α) more than doubles tritiated thymidine ([3H]TdR) uptake in mouse macrophages stimulated by macrophage colony‐ stimulating factor (CSF‐1). However, nothing is known of how TNF‐α affects this increase or even whether it is manifested by increased cellular proliferation. Here we characterize the effects of TNF‐α on CSF‐l‐stimulated proliferation of both primary cells (bone marrow‐derived macrophages, BMMs) and a cloned growth factor‐dependent macrophage cell line (SI). We show that the TNF‐ α‐induced increase in [3H]TdR uptake of CSF‐l‐stimu‐ lated macrophages is directly proportional to an increase in the DNA content of the culture and that the effects of TNF‐α are direct and independent of cell number. TNF‐a decreases the population doubling time of log‐phase growing macrophages having quite different growth rates to the same (approximately 30%) extent: the doubling time of BMMs decreases from 24 to 17 h and that of SI cells from 17 to 13 h. TNF‐α exerts its effects on log‐phase growth by increasing to the same proportion CSF‐l‐stimulated proliferation at all concentrations of CSF‐1; that is, TNF‐α does not shift, but rather amplifies, the CSF‐1 dose‐response curve. Although TNF‐α alone does not stimulate macrophage proliferation, its presence in S1 cell cultures coming to quiescence after withdrawal of CSF‐1 greatly increases subsequent CSF‐l‐stimulated [3H]TdR uptake as the cells reenter the cycle. Finally, we show that both human and mouse TNF‐α increase CSF‐l‐stimulated log‐phase growth and reentry of quiescent cells into the cycle equally on a molar basis (halfmaximal stimulation of approximately 0.3 nM). The latter observation argues that the growth‐stimulatory effects of TNF‐α are mediated via the 55‐60‐kd TNF receptor. We conclude that TNF‐α acts directly on growth‐ competent macrophages to decrease significantly the population doubling time in a manner that enhances the mitogenic effects of CSF‐1.

Caspase activation is not required for villous cytotrophoblast fusion into syncytiotrophoblasts

The villous trophoblast renews itself by fusion of individual stem cells (cytotrophoblasts, CT) with a functional syncytium (syncytiotrophoblast, ST). The literature indicates that fusion occurs with limited activation (proteolytic cleavage) of caspase-8 in CT and is inhibited either by blocking caspase-8 synthesis or inhibiting activation with a caspase-8-specific inhibitor, zIETD. We challenge part of this evidence: inhibition of differentiation with caspase-8 inhibitors. Br-cAMP-stimulated differentiation of isolated CT into multinucleated syncytia in culture is not blocked with three different low molecular weight inhibitors of caspase-8: broad caspase inhibitors zVAD-fmk and qVD-OPh and the caspase-8-specific inhibitor zIETD-fmk. Syncytialization was determined by desmoplakin staining of intracellular boundaries surrounding >2 nuclei and by diffusion within fused cells of long-lived cytoplasmic staining from half of original CT to the unstained half. Differentiation of isolated CT into hCGβ-secreting syncytiotrophoblast was also not blocked by the inhibitors nor was upregulation of hCGβ secretion blocked in ST-stripped and regenerated 5 day explant cultures. The ratio of CT to ST nuclei present was also not changed in explant cultures by caspase inhibitors. The effectiveness of caspase inhibitors was demonstrated by their ability to completely block TNFα-induced apoptosis. We conclude that activation of caspases in general, and caspase-8 in particular, is not required for villous CT differentiation into ST. However, another role of intact caspase-8 (proform) in CT differentiation remains possible.

Pleiotropic Actions of Forskolin Result in Phosphatidylserine Exposure in Primary Trophoblasts

Forskolin is an extract of the Coleus forskholii plant that is widely used in cell physiology to raise intracellular cAMP levels. In the field of trophoblast biology, forskolin is one of the primary treatments used to induce trophoblastic cellular fusion. The syncytiotrophoblast (ST) is a continuous multinucleated cell in the human placenta that separates maternal from fetal circulations and can only expand by fusion with its stem cell, the cytotrophoblast (CT). Functional investigation of any aspect of ST physiology requires in vitro differentiation of CT and de novo ST formation, thus selecting the most appropriate differentiation agent for the hypothesis being investigated is necessary as well as addressing potential off-target effects. Previous studies, using forskolin to induce fusion in trophoblastic cell lines, identified phosphatidylserine (PS) externalization to be essential for trophoblast fusion and showed that widespread PS externalization is present even after fusion has been achieved. PS is a membrane phospholipid that is primarily localized to the inner-membrane leaflet. Externalization of PS is a hallmark of early apoptosis and is involved in cellular fusion of myocytes and macrophages. We were interested to examine whether PS externalization was also involved in primary trophoblast fusion. We show widespread PS externalization occurs after 72 hours when fusion was stimulated with forskolin, but not when stimulated with the cell permeant cAMP analog Br-cAMP. Using a forskolin analog, 1,9-dideoxyforskolin, which stimulates membrane transporters but not adenylate cyclase, we found that widespread PS externalization required both increased intracellular cAMP levels and stimulation of membrane transporters. Treatment of primary trophoblasts with Br-cAMP alone did not result in widespread PS externalization despite high levels of cellular fusion. Thus, we concluded that widespread PS externalization is independent of trophoblast fusion and, importantly, provide evidence that the common differentiation agent forskolin has previously unappreciated pleiotropic effects on trophoblastic cells.

The characterization of fibrocyte-like cells: A novel fibroblastic cell of the placenta

The placenta is a highly vascularized organ thus angiogenesis is a key process in placental development. The contribution that different cells in the villous stroma play in placental angiogenesis is largely unknown. In this study we identified a novel stromal cell type in sections of term placenta which is morphologically fibroblastic and expressing the fibroblast marker TE-7 but also positive for the monocytic markers CD115 and CD14 and designated these cells as fibrocyte-like cells. Populations of fibrocyte-like cells from the placenta were isolated by two methods: culture of adherence-selected placental cells and, for higher purity, by CD45 fluorescence activated cell sorting (FACS). Fibrocyte-like cell conditioned medium increased endothelial tubule-like structure formation 2-fold versus control medium. Both pro-angiogenic growth factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) and the anti-angiogenic factor soluble-Flt were found in the conditioned medium. Neutralizing antibodies against VEGF and b-FGF reduced endothelial cell tubule-like structures to control levels. These data suggests that fibrocyte-like cells, a previously unidentified cell of the villous stroma, may play an important role in the regulation of placental angiogenesis.

The contribution of apoptosis-inducing factor (AIF) to villous trophoblast differentiation

Apoptosis is postulated to be a delayed but important part of the differentiation of placental villous cytotrophoblasts (CT) into functional syncytiotrophoblast (ST). This hypothesis is based on the observation that the externalization of phosphatidylserine and the activation of caspase 8 are required for trophoblast differentiation. In contradiction to this hypothesis we have previously found that differentiation occurs in the presence of both broad spectrum and caspase 8 specific inhibitors. Apoptosis-inducing factor (AIF) is a mitochondria-associated protein which is known to translocate to the nucleus and induce caspase-independent nuclear condensation, phosphatidylserine externalization and cell death. Thus AIF nuclear translocation may result in the apoptotic-like features associated with trophoblast differentiation and may be an obligatory event for differentiation to proceed. AIF translocation was assessed in isolated primary trophoblasts by optical section microscopy of antibody stained cells. We found AIF to be strongly expressed in the villous trophoblast and that small amounts of AIF were localized to the nucleus of the cells. Significantly, inhibitors of AIF translocation (calpeptin and zFA-fmk) blocked translocation but not differentiation of the cells. We conclude that AIF translocation is not involved in CT differentiation in isolated cell culture.