Extravillous trophoblast (EVT) cells of the human placenta invade the uterine decidua and utero-placental arteries to establish an efficient exchange of key molecules between maternal and fetal blood. Trophoblast invasion is stringently regulated in situ both positively and negatively by a variety of factors at the fetal-maternal interface to maintain a healthy utero-placental homeostasis. One such factor, decorin, a transforming growth factor (TGF)-beta binding, leucine-rich proteoglycan produced by the decidua, negatively regulates EVT proliferation, migration, and invasiveness independent of TGF-beta. We reported that these decorin actions were mediated by its binding to multiple tyrosine kinase receptors, including vascular endothelial growth factor receptor (VEGFR)-2. The present study explores the mechanisms underlying decorin antagonism of VEGF (VEGF-A) stimulation of endovascular differentiation of EVT using our EVT cell line, HTR-8/SVneo. We observe that decorin inhibits VEGF-induced EVT cell migration and endothelial-like tube formation on matrigel. VEGF activates MAPKs (p38 MAPK, MEK3/6, and ERK1/2) in EVT cells, and the activation is blocked in both cases by decorin. Employing selective MAPK inhibitors, we show that both p38 and ERK pathways contribute independently to VEGF-induced EVT migration and capillary-like tube formation. VEGF upregulates the vascular endothelial (VE) markers VE-cadherin and beta-catenin in EVT and endothelial cells, and this upregulation is blocked by decorin and MAPK inhibitors. These results suggest that decorin inhibits VEGF-A stimulation of trophoblast migration and endovascular differentiation by interfering with p38 MAPK and ERK1/2 activation. Thus decorin-mediated dual impediment of endovascular differentiation of the EVT and angiogenesis may have implications for pathogenesis of preeclampsia, a hypoinvasive trophoblast disorder in pregnancy.

Perianal ulcers in an otherwise healthy individual is an unusual presentation for perforating disorders but should be considered in cases not responding to traditional treatment options.

The human placenta is a highly invasive organ that invades the pregnant uterus to nourish the fetus. It develops from highly proliferative and phenotypically plastic cells called trophoblasts. Bi-potential trophoblast stem cells in the chorionic villi differentiate into the villous pathway to form the syncytiotrophoblast layer and the extravillous trophoblast (EVT). The HTR-8/SVneo cell line is widely used to study trophoblast biology. These cells simultaneously express villous-specific genes such as hCG and EVT-specific genes such as VE-Cadherin. Such phenotypic plasticity is indicative of a bi-potential cytotrophoblast progenitor. Preliminary work has shown that similar to the progenitors in situ, a subpopulation of HTR-8/SVneo cells expresses α6β4 integrin. This cell line also expresses Nodal, a stem cell-associated factor that sustains the pluripotency of embryonic stem cells and is re-expressed in certain cancers. We hypothesize that an α6β4high subset within HTR-8/SVneo cell line is enriched with bi-potential progenitors and that the stem cell-like properties of this population is maintained by Nodal signaling. Immunofluorescence microscopy and flow cytometry were used to confirm the presence of an α6β4high subpopulation in the HTR-8/SVneo cell line. Fluorescence Activated Cell Sorting (FACS) was used to purify α6β4high and α6β4low populations from the heterogeneous HTR-8/SVneo cells. The ability of α6β4high, α6β4low and heterogeneous (unsorted) HTR-8/SVneo populations to undergo self-renewal was determined using a limiting dilution clonogenic assay. Differentiation into endovascular-like trophoblast was assessed using a tube-formation assay on Matrigel and invasion assays, and differentiation into villous trophoblast was assessed by measuring α-hCG. The expression of Nodal in the α6β4 subtypes was assayed using Western blotting. We determined that approximately 25% of HTR-8/SVneo cells express α6β4 integrin protein on their cell surface. The α6β4high subpopulation had a significantly higher self-renewal capacity than the heterogeneous HTR-8/SVneo population and the α6β4low subpopulation had a lower clonogenic capacity relative to the α6β4high and heterogeneous cells. Nearly 100% of the α6β4high cells formed tubes when plated on Matrigel. In contrast, the unsorted HTR-8/SVneo cells formed tubes much less efficiently, and the α6β4low population was unable to undergo tube formation. The α6β4high population was also more invasive than the α6β4low cells, and Nodal was expressed to a greater level in the α6β4high population. Conclusions: Our results suggest that α6β4high sub-population in the HTR-8SV/neo cell line may represent a villous cytotrophoblast stem cell and that this subpopulation is enriched with Nodal. By understanding the mechanisms by which this subpopulation is regulated, we may glean insight into the regulation of invasive cancer stem cell populations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3354. doi:1538-7445.AM2012-3354