Merete Wiiger

Ex vivo and In vivo Delivery of Anti-Tissue Factor Short Interfering RNA Inhibits Mouse Pulmonary Metastasis of B16 Melanoma Cells

Purpose: The coagulation trigger tissue factor has been implicated in tumor growth, angiogenesis, and metastasis. In this study, we explore the effects of ex vivo and in vivo delivery of short interfering RNA (siRNA) targeting tissue factor on B16 melanoma colonization of the lung in a murine model for metastasis. The purposes of this work are to establish a noncytotoxic in vivo model for investigation of tissue factor function and provide preclinical assessment of the therapeutic potential of tissue factor siRNA for prevention of metastasis. Experimental Design and Results: C57BL/6 mice were evaluated for pulmonary metastases following tail vein injection of B16 cells transfected with either active or inactive siRNA. Mice receiving cells transfected with active siRNA had significantly lower numbers of pulmonary tumors compared with mice injected with control cells (transfected with inactive siRNA). The average time point at which the mice started to exhibit tumor-associated stress was also increased significantly from 22 days for the control group to 27 days for the experimental group (P = 0.01). In a therapeutically more relevant model, where the siRNA was delivered i.p. and the cells (untransfected) by tail vein injection, an inhibitory effect on metastasis was observed when the siRNA treatment was initiated either before or at the time of cell injection. Conclusions: The results suggest that tissue factor has a crucial function in promoting lung tumor metastasis of blood-borne tumor cells in the early stages of the tumor take process and further suggest that treatment with tissue factor siRNA may become a viable clinical strategy for prevention of tumor metastasis.

Polar expression of tissue factor in human umbilical vein endothelial cells

Endothelial cells grown on filters developed junctional complexes that reduced diffusional transport and increased electrical resistance over the cell layer. Induction of tissue factor by recombinant interleukin-1 beta led to a highly polarized tissue factor expression on the apical cell surface only. After prolonged growth to allow deposition of matrix, removal of the endothelial cells by collagenase or by 0.1 mol/L NH4OH left behind some cellular material as well as tissue factor, which was only detectable in the upper compartment. A human bladder carcinoma cell line, which does not form tight junctions and expresses tissue factor constitutively, showed essentially no polarity. Endothelial cell secretory compounds like von Willebrand factor, tissue plasminogen activator, and plasminogen activator inhibitor-1 were constitutively released to both sides. The added secretion due to recombinant interleukin-1 beta stimulation of the endothelial cells observed for von Willebrand factor and tissue plasminogen activator was, however, localized to the apical surface. The availability of tissue factor on the luminal surface of endothelial cells, ie, allowing contact with factor VII in the flowing blood, has potentially very significant pathophysiological consequences.

The epidermal growth factor receptor (EGFR) and proline rich tyrosine kinase 2 (PYK2) are involved in tissue factor dependent factor VIIa signalling in HaCaT cells

Binding of the coagulation protease factor VIIa to its receptor Tissue Factor (TF) induces intracellular signals in several cell types including HaCaT keratinocytes. TF belongs to the cytokine receptor family, but is most likely not alone in transferring the complete TF/FVIIa signal over the plasma membrane. The protease activated receptor PAR2 is involved in factor VIIa and factor Xa signal transduction. Our results indicate that the epidermal growth factor receptor (EGFR) and the proline rich tyrosine kinase 2 (PYK2) participate in TF/FVIIa signalling as formation of the TF/FVIIa complex increased the phosphorylation of these proteins. Both FVIIa protease activity and available TF were necessary for generation of the signal. Increased tyrosine phosphorylation of the EGFR was observed following TF/FVIIa complex formation on the cell surface. The EGFR kinase inhibitor tyrphostin AG1478 abrogated the TF/FVIIa-complex induced MAP kinase activation and mRNA increase of egr-1, heparin-binding EGF, and interleukin-8 following FVIIa addition. Using specific antibodies, increased phosphorylation of PYK2 tyrosine residues 402 and 580 was observed. The first site is the major autophosphorylation site and the docking site for Src family kinases. The second site is important for the kinase activity. The Src family kinase Yes and the tyrosine phosphatase SHP-2 were detected in immunoprecipitates using either anti-PYK2 or anti-EGFR antibodies. Their coprecipitation with EGFR increased in the presence of FVIIa. Moreover, the coprecipitation of EGFR and PYK2 increased with FVIIa stimulation. Together, these data suggest that EGFR, PYK2, Yes, and SHP-2 are involved in transduction of the TF/FVIIa signal possibly via transactivation of the EGF receptor.

Cellular Effects of Initiation of the Extrinsic Pathway of Blood Coagulation

In the initial phase of scientific research into blood clotting around 50 years ago, most studies focused on investigating blood samples to find out what took place in the flowing blood. With the purification and cloning of Tissue Factor (TF) it was realized that TF was an integral membrane protein sitting in the cell surface membrane. This shifted the emphasis to investigations of what happened on the cell surface, and later to the cell biology of TF and its inducibility in monocytes/macrophages and endothelial cells. During the last 8 years, researchers have become increasingly interested in studying the processes going on inside the cells that carry TF when coagulation is initiated on their surface. Cells carrying TF have been incriminated in tumorigenesis, metastasis, angiogenesis, and a number of other cellular phenotypes. That binding of the plasma clotting Factor VIIa upregulates a number of genes involved in regulation of growth, transcription, and cellular motility, as well as cytokines, makes it possible to suggest a link between the formation of the TF/Factor VIIa complex and these cellular processes.

The changing faces of tissue factor biology: A personal tribute to the understanding of the "extrinsic coagulation activation"

The changing faces of tissue factor biology - A personal tribute to the understanding of the “extrinsic coagulation activation” -

Effects of Binding of Ligand (FVIIa) to Induced Tissue Factor in Human Endothelial Cells

The tissue factor protein is structurally related to the cytokine receptors and ligand binding (factor VIIa) has been reported to give an intracellular calcium signal, thus indicating that tissue factor is a true receptor. In view of the attempts to use recombinant factor VIIa as a therapeutic agent in hemophilia, its binding effects may be of clinical interest. We have studied the effect of ligand binding to human endothelial cells that were stimulated with interleukin-1 to express tissue factor. Human umbilical cord vein endothelial cells produce and release a wide variety of proteins that participate in coagulation and fibrinolysis, and we have investigated whether binding of recombinant factor VIIa to tissue factor altered the release of some of these compounds. Three main findings are reported. (1) After an initial increase, the measurable tissue factor activity in endothelial cells decreased more rapidly in the presence of factor VIIa (half-life 3.7+/-0.7 hours) than in its absence (half-life 7.4+/-1.5 hours). This difference was not seen when tissue factor antigen was measured, indicating that ligand binding did not increase the degradation of the protein. (2) Tissue factor pathway inhibitor was detected on the cell surface, in cell homogenates, and in cell medium. When recombinant factor VIIa was added to the cells there was a significant decrease in the release of tissue factor pathway inhibitor to the medium. Four hours after recombinant factor VIIa was added, the levels were 7.5-fold higher in the medium of untreated cells compared to the medium of cells treated with recombinant factor VIIa. (3) We observed increased release of von Willebrand factor (vWF). After 1 and 6 hours with recombinant FVIIa the release was significantly greater than in controls without FVIIa. We did not detect significant differences in the release of tissue plasminogen activator or tissue factor pathway inhibitor.

A novel gene mutation in the 60s loop of human coagulation factor VII - inhibition of interdomain crosstalk.

A novel mutation in the factor VII gene resulting in procoagulant activity of 7.5% and antigen levels of 23% is presented. Single-stranded conformational polymorphism and DNA sequencing analysis revealed heterozygous shifts, and mutations were detected in exons 5, 7 and 8. The mutant L204P in exon 7 was novel, while the common polymorphisms, H115H and R353Q, were located in exons 5 and 8, respectively. The molecular effect of the L204P mutation was characterized using recombinant mammalian expression in Chinese hamster ovary cells. Low levels (4 ng/ml) of secreted mutant protein were found in transiently transfected cells compared to wild-type factor VII (83 ng/ml). Metabolic labeling demonstrated that the rate of mutant protein synthesis was similar to that of wild-type FVII, and the mutant protein accumulated intracellularly with no signs of increased degradation during a four-hour chase. No interaction between secreted P204 protein and immobilized soluble tissue factor was detected using surface plasmon reso-nance. The activation rate of recombinant mutant FVII protein was strongly reduced compared to wild-type FVII. A 9-fold reduction in the rate of FX activation was detected whereas Km was nearly the same for wild-type and the mutant. This slow rate was caused by a correspondingly lowered rate of P204 activation. A synthetic peptide sequence comprising amino acids 177-206 blocked binding of FVIIa to the TF-chip, and the subsequent factor X activation with an IC(50) value of 0.5 micro M in a chromogenic factor Xa assay. Additionally, evaluation of the peptide by surface plasmon resonance analysis resulted in inhibition of complex formation with an apparent K(I) of 7 micro M.