These data suggest that the trophoblast-derived anti-angiogenic molecule PEDF is involved in restricting growth and expansion of the feto-placental endothelium predominantly in late pregnancy and focuses on to modulate the intracellular effect of VEGF. Electronic supplementary material The online version of this article (doi:10.1007/s10456-016-9513-x) contains supplementary material, which is available to authorized users. absent Primary 1st trimester trophoblast cells (FTB) 1st trimester villous trophoblasts were isolated (to remove deceased cells and cell debris. late pregnancy trophoblast. Notably, human being recombinant PEDF reduced network formation only in combination with VEGF. Also in the CAM assay, the combination of PEDF with VEGF reduced branching of vessels below control levels. Analysis of phosphorylation of ERK1/2 and FAK, two important players in VEGF-induced proliferation and migration, exposed that PEDF modified VEGF signaling, while PEDF only did not impact phosphorylation of ERK1/2 and FAK. These data suggest that the trophoblast-derived anti-angiogenic molecule PEDF is definitely involved in restricting growth and expansion of the feto-placental endothelium mainly in late pregnancy and focuses on to modulate the intracellular effect of VEGF. Electronic supplementary material The online version of this article (doi:10.1007/s10456-016-9513-x) contains supplementary material, which is available to authorized users. absent Main 1st trimester trophoblast cells (FTB) First trimester villous trophoblasts were isolated (to remove deceased cells and cell debris. CM was aliquoted BOP sodium salt and stored at ?80?C. CM was pooled to enable comparable screening with numerous assays using the same CM pool. At least two swimming pools of 1st and third trimester trophoblast from two to four different isolations were used. Like a control (non-CM), DMEM/EBM with 7.5?% FBS was incubated at the same conditions. In vitro network formation assay To observe network formation, 1??104 feto-placental endothelial cells were resuspended in conditioned/treatment medium and plated on growth factor-reduced Matrigel (BD Bioscience, USA). Tube-like constructions were visualized after 12-h incubation by a Zeiss Cell Observer microscope with an AxioCam HRm video camera and an A-Plan 5x/0.12 Ph0 objective using the software AxioVision (Carl Zeiss Imaging Solutions GmbH). For quantification the total tube size, the branching points and the number of meshes were analyzed from the ImageJ software (NIH) using the AngioJ-Matrigel assay plugin, kindly provided by Diego Guidolin (Division of Human Anatomy and Physiology, Section of Anatomy, University or college of Padova, Italy) . Therefore, total network size, quantity of branching points and meshes were counted. As representative parameter total tube length can be used because branching points and quantity of meshes show the same tendency. Migration/chemoattraction assay Migration/chemoattraction of medium was observed using a 96-well chemotaxis microplate system (Neuro Probe Inc, UK). After serum starvation for 3?h in EBM, 1??104 cells per well were placed in the upper part of the chemotaxis system, which was separated from the lower well by a fibronectin-coated polycarbonate filter with 8-m pores. Cells were allowed to migrate toward chemoattractants in the lower well (CM) for 4?h at 37?C. As positive control, DE medium supplemented with FBS and growth factors (EGM-MV BulletKit, Lonza) was used. The upper surface of the filter was wiped clean of non-migrating cells. Cells were fixed with 4?% formaldehyde and stained with DAPI (Invitrogen, USA). Subsequently, the microplate was observed by a Zeiss Axioplan fluorescence microscope and a 10 objective using the AxioVision software (Carl Zeiss Imaging Solutions GmbH). From each filter well 35 photos were taken. Out of these, 7 photos were randomly selected and analyzed using DotCount v1.2 (online provided by Martin Reuter, MIT). Proliferation assay Proliferation of feto-placental endothelial cells was assessed using the BrdU ELISA kit (Cyclex, Japan) according to the manufacturers recommendations. 6??103 cells per well were seeded inside a 96-well plate. After 24?h, the medium was changed to the conditioned/treatment medium and cells were incubated for another 24?h. Subsequently, BrdU was added to a final concentration of 10?M and incubated for 2?h. Cells were fixed, denaturized and incubated with the monoclonal antibody against BrdU. Absorbance was measured immediately at 450/540?nm using the FluoSTAR Optima 413 spectrofluorometer (BMG Lab systems, Germany). LDH assay Cytotoxicity of conditioned/treatment medium on feto-placental endothelial cells was tested by measurement of released lactate dehydrogenase (LDH, Takara, Japan) according to the manufacturers instructions. 6??103 cells per well were seeded inside a 96-well plate with the conditioned/treatment medium for 24?h. Absorbance was measured immediately at 490/650?nm using the Spectromax 250 molecular products microplate reader (MWG-Biotech, Germany). Chick chorioallantoic membrane (CAM) assay To determine the effect of CM on angiogenesis, the ex lover ovo chorioallantoic membrane (CAM) assay was performed. Briefly, fertilized white leghorn chicken (L.) eggs (Schropper GmbH, Gloggnitz, Austria) were incubated for 3?days at 37.6?C and 70C75?% relative moisture (J. Hemel Mouse monoclonal to Cytokeratin 5 Brutger?te, Am Buschbach, Germany). Eggs were then opened into plastic weigh boats covered with square Petri dishes and returned to the incubator. On day time ten, six on-plants were placed on the CAM vasculature. The on-plants consisted of a silicone ring comprising either FTB CM, TTB CM or non-conditioned control medium, each on four different eggs. On day time 3, BOP sodium salt vascularization of the on-plants was obtained by a blinded BOP sodium salt observer using.