Demonstration of a Tissue Factor Pathway Inhibitor 2 Messenger RNA Synthesis by Pure Villous Cytotrophoblast Cells Isolated from Term Human Placentas1

Abstract

Tissue factor pathway inhibitor 2 (TFPI-2), a Kunitz-type proteinase inhibitor, might play an important role during placenta growth by regulating trophoblast invasion and differentiation. Many TFPI-2 transcripts have been detected in syncytiotrophoblast cells, but conflicting results have been reported concerning TFPI-2 synthesis by the cytotrophoblast. To address this issue, we developed a method to isolate pure preparations of human villous cytotrophoblast cells from normal term placentas, and the synthesis of tissue factor, TFPI-1, and TFPI-2 mRNAs was then evaluated. Cells were isolated by trypsin-DNase-EDTA digestion, followed by Percoll gradient separation and immunodepletion of human leukocyte antigen-positive cells. The quality of villous cytotrophoblast cells was verified by electron microscopy. Purity of cell preparations was assessed by labeling cells with GB25, a monoclonal antibody specific to villous trophoblast cells, and by checking the absence of contaminating cells using anti-CD9 antibody. The lack of hCG, CD32 mRNA, and tissue factor mRNA also indicated the absence of contaminating cells. Using competitive reverse transcription polymerase chain reaction, we showed that freshly isolated villous cytotrophoblast cells synthesized significant levels of TFPI-1 mRNA and larger amounts of TFPI-2 mRNA. TFPI-1 and TFPI-2 mRNA synthesis remained unchanged when cytotrophoblast cells were cultured in complete medium and evolved as a multinucleated syncytiotrophoblast. These results indicate that the villous cytotrophoblast and syncytiotrophoblast are both important sites of TFPI-2 synthesis in the human placenta. This study also indicates that tissue factor detection should be used systematically to check the purity of cytotrophoblast cell preparations because it allows detection of contamination by monocytes/macrophages and by syncytial fragments.

A link between a hemostatic disorder and preterm PROM: a role for tissue factor and tissue factor pathway inhibitor

“…TFPI-2, first isolated from human placenta as Placental Protein 5 (PP5) [32,33], is the major form of TFPI in the placenta [26-31], and it is expressed by the syncytiotrophoblast [35,36]. In the non-pregnant state, TFPI-2 is present mainly in the extracellular matrix and its plasma concentrations are very low [33-35].…”

“…The mean maternal plasma concentration of total TFPI (TFPI-1 and TFPI-2) have been reported to increase during pregnancy until 20 weeks of gestation, to remain relatively constant until term [22] and to decrease during labor [23]. There are two types of TFPI: 1) TFPI-1 is the more prevalent form in the non-pregnant state in the maternal circulation and can also be found in the fetal blood, platelets, endothelial cells and other organs [24,25]; and 2) TFPI-2- the major form of TFPI in the placenta [26-31], is also known as Placental Protein 5 (PP5) [32,33]. During pregnancy, the maternal plasma concentration of TFPI-2 increases gradually, reaches a plateau at 36 weeks and subsides after delivery [33-38].…”

Expression and methylation status of tissue factor pathway inhibitor-2 gene in non-small-cell lung cancer

“…Three clones were selected after each experiment and sequenced to determine the methylation status in every sample. Choriocarcinoma JAR cells and trophoblast cells isolated from normal placentas (Hube et al, 2003c) were also studied as positive (i.e. hypermethylated) and negative controls for every experiment (RE-PCR or bisulphite genomic sequencing).…”

The interaction of the second Kunitz-type domain (KD2) of TFPI-2 with a novel interaction partner, prosaposin, mediates the inhibition of the invasion and migration of human fibrosarcoma cells

“…TFPI-2 is secreted into the ECM (extracellular matrix) by a wide variety of human cells, including endothelial cells [1], monocytes [2], fibroblasts [3], epithelial cells [4], smooth muscle cells [5], syncytiotrophoblast cells [6,7], and several human tumour cells [8][9][10][11]. TFPI-2 is secreted into the ECM (extracellular matrix) by a wide variety of human cells, including endothelial cells [1], monocytes [2], fibroblasts [3], epithelial cells [4], smooth muscle cells [5], syncytiotrophoblast cells [6,7], and several human tumour cells [8][9][10][11].…”