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PO-09 Expression of alternatively spliced tissue factor in human endothelial cells: induction by inflammatory stimuli and modulation by heparins
Abstracts / Thrombosis Research 120 Suppl. 2 (2007) S145–S178 present further evidence for the role of TFPI in the proliferation process. PO-07 Dual implication of endothelial protein C receptor (EPCR) in cancer homoeostasis E. Ducros1 *, S.S. Mirshahi2 , J.Y. Perrot3 , A.M. Faussat1 , L. Chauvenet3 , F. Daggonet4 , E. Pujade-Lauraine3 , J. Soria1 , M. Mirshahi1 . 1 CRC, Facult´ e de M´ edecine Paris VI; 2 Stago R&D, otel Dieu, Paris; 4 Laboratoire Gennevilliers; 3 Service d’Oncologie, Hˆ Biotechnologie et Oeil, Facult´ e Paris V, Paris, France Introduction: At the cell surface, thrombin enhances adhesion between tumor cells, platelets, inflammatory cells, ECs, ECM and contributes to tumor progression. Activated Protein C (APC) is generated through interaction with EPCR and IIa/thrombomodulin receptor complex. APC inactivates factors Va and VIIIa to provide negative feedback to thrombin generation and to avoid disseminated coagulation. Aim: To study the expression of EPCR in several cancers and its role in promoting tumor cell progression, regulation of pro-coagulant activity and ascites formation. Materials and Methods: Cellular clusters were isolated from various cancer patients’ ascitic fluids. Electronic microscopy and FACS analysis were performed to identify cellular clusters constituents. EPCR and TF expression were studied by RT-PCR, immunocytochemistry and ELISA. Hemostasic status of ascitic fluids were evaluated by measuring modification of partial thromboplastin clotting time (APTT) of normal plasma induced by ascites. Results: With cancer cells, clusters were composed of B, T and NK lymphocytes, monocytes and dendritic cells and were mostly aggregate with fibrin fibers. Ascitic fluids supernatants mainly show procoagulant and anticoagulant activity, provided by TF and soluble EPCR respectively. However, EPCR was identified in cellular clusters. Conclusion: Beyond hemostasis, other functions related to inflammation, proliferation and apoptosis have been attributed to APC, APC/EPCR interaction on cancer cell surface (1) provides protection for tumor cell through its antiapoptotic effect, (2) inhibits ascitic “cancer inflammatory cells clusters” procoagulant activity by downregulation of thrombin generation and (3) promotes cancer cell progression and (4) ascite formation. PO-08 Defibrotide, an anti-thrombotic drug, prevents angiogenesis in vitro and in vivo G. Eissner1,2 *, G.E. Koehl3 , E.G. Geissler3 , E. Holler2 , M. Iacobelli1 . Sp.A., Villa Guardia (CO), Italy, 2 Department of Hematology and Oncology, and 3 Department of Surgery, Regensburg University Medical Center, Regensburg, Germany 1 Gentium,
Defibrotide (DF) is a polydisperse mixture of 90% single-stranded polydeoxyribonucleotides with anti-thrombotic and anti-ischemic functions. Among other indications, this drug is used in the treatment of veno-occlusive disease following allogeneic hematopoetic stem cell transplantation. DF has anti-inflammatory activity through preventing transendothelial migration of immune effector cells and their alloreactivity towards the endothelium. In addition, we have provided evidence that DF can protect endothelial cells from chemotherapy-mediated apoptosis, suggesting its prophylactic use in patients at risk for endothelial complications. Next to these endothelium stabilizing functions, recent preclinical evidence suggests that DF might also have anti-neoplastic properties. We addressed the question whether this might be due to the prevention of tumor angiogenesis. The anti-angiogenic potential of DF was tested in vitro using human microvascular endothelial cells forming vessel structures across a layer of dermal fibroblasts (AngioKitTM ). Our results demonstrate that DF at concentrations corresponding to pharmacologic DF blood levels
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reduces vessel formation, when applied on a daily basis. This result could be confirmed by two alternative in vitro angiogenesis assays (MatrigelTM , rat aortic ring assay). In vivo, tumor angiogenesis in the murine dorsal skin-fold chamber model using the inoculation of human gastric cancer (TMK-1) cells was also attenuated by DF. Regarding the signal transduction mechanism of DF, Western blotting results with activation-specific antibodies show that DF reduces phosphorylation-activation of p70S6 kinase, which is a key component of the PI3K/Akt/mTOR signaling pathway linked to angiogensis. This could be confirmed in a cell-free mTOR K-LISA assay. In addition, DF acts independent of a blockade of vascular endothelial growth factor (VEGF), suggesting the involvement of other angiogenic factors. Taken together, our data suggest that while DF is known for its endothelium-protecting function, it also inhibits (tumor) blood vessel formation, presumably via normalization and/or migration inhibition of endothelial cells, and thus should be considered for further testing as an anticancer agent, either alone or in combination with other drugs. PO-09 Expression of alternatively spliced tissue factor in human endothelial cells: induction by inflammatory stimuli and modulation by heparins M. Brambilla1 , D. Colnago2 , A. Pignieri1 , L. Mussoni1 , E. Tremoli1,2 , M. Camera1,2 *. 1 Dept. Pharmacological Sciences, University of Milan; 2 Centro Cardiologico Monzino, Milan, Italy Introduction: Tissue factor (TF), the principal initiator of blood coagulation, also contributes to angiogenesis and metastasis. Under physiological conditions, TF is absent from quiescent endothelium but it can be induced by inflammatory stimuli. In malignant tissue, TF is present in neoplastic cells as well as endothelial cells. Recently an alternatively spliced form of TF (asTF) has been identified, which lacks exon 5 and is therefore soluble. Although heparin has been reported to be able to modulate TF expression, no information is still available on the pharmacological modulation of asTF in endothelial cells. Aim: To investigate the kinetic of asTF mRNA expression and the modulation of TF and asTF by unfractionated (UH) and low molecular weight heparin (LMWH). Methods: Endothelial cells (HUVEC) were incubated with heparins (0.001 10 UI/ml) and stimulated with LPS (1 mg/ml) or TNF-a (10 ng/ml). TF and asTF mRNA levels were quantified by Real Time PCR. TF pro-coagulant activity was also evaluated. Results: Stimulation of HUVEC with TNF-a resulted in a timedependent increase of both TF and asTF mRNA levels, with a peak of effect after 60 minutes stimulation for both transcripts. Treatment with UH or LMWH resulted in a concentration-dependent reduction of TF and asTF mRNA levels ( 40% and 30% respectively with the highest concentration). Both heparins were also able to significantly reduce a concentration dependent manner TNF-a and LPS-induced pro-coagulant activity. Conclusion: TNF-a and LPS stimulation of HUVEC induces a concomitantly upregulation of TF and asTF mRNA. The heparininduced down-regulation of asTF mRNA levels might further strengthen the antithrombotic effect of this drug.
Defibrotide (DF) is a polydisperse mixture of 90% single-stranded polydeoxyribonucleotides with anti-thrombotic and anti-ischemic functions. Among other indications, this drug is used in the treatment of veno-occlusive disease following allogeneic hematopoetic stem cell transplantation. DF has anti-inflammatory activity through preventing transendothelial migration of immune effector cells and their alloreactivity towards the endothelium. In addition, we have provided evidence that DF can protect endothelial cells from chemotherapy-mediated apoptosis, suggesting its prophylactic use in patients at risk for endothelial complications. Next to these endothelium stabilizing functions, recent preclinical evidence suggests that DF might also have anti-neoplastic properties. We addressed the question whether this might be due to the prevention of tumor angiogenesis. The anti-angiogenic potential of DF was tested in vitro using human microvascular endothelial cells forming vessel structures across a layer of dermal fibroblasts (AngioKitTM ). Our results demonstrate that DF at concentrations corresponding to pharmacologic DF blood levels
S147
reduces vessel formation, when applied on a daily basis. This result could be confirmed by two alternative in vitro angiogenesis assays (MatrigelTM , rat aortic ring assay). In vivo, tumor angiogenesis in the murine dorsal skin-fold chamber model using the inoculation of human gastric cancer (TMK-1) cells was also attenuated by DF. Regarding the signal transduction mechanism of DF, Western blotting results with activation-specific antibodies show that DF reduces phosphorylation-activation of p70S6 kinase, which is a key component of the PI3K/Akt/mTOR signaling pathway linked to angiogensis. This could be confirmed in a cell-free mTOR K-LISA assay. In addition, DF acts independent of a blockade of vascular endothelial growth factor (VEGF), suggesting the involvement of other angiogenic factors. Taken together, our data suggest that while DF is known for its endothelium-protecting function, it also inhibits (tumor) blood vessel formation, presumably via normalization and/or migration inhibition of endothelial cells, and thus should be considered for further testing as an anticancer agent, either alone or in combination with other drugs. PO-09 Expression of alternatively spliced tissue factor in human endothelial cells: induction by inflammatory stimuli and modulation by heparins M. Brambilla1 , D. Colnago2 , A. Pignieri1 , L. Mussoni1 , E. Tremoli1,2 , M. Camera1,2 *. 1 Dept. Pharmacological Sciences, University of Milan; 2 Centro Cardiologico Monzino, Milan, Italy Introduction: Tissue factor (TF), the principal initiator of blood coagulation, also contributes to angiogenesis and metastasis. Under physiological conditions, TF is absent from quiescent endothelium but it can be induced by inflammatory stimuli. In malignant tissue, TF is present in neoplastic cells as well as endothelial cells. Recently an alternatively spliced form of TF (asTF) has been identified, which lacks exon 5 and is therefore soluble. Although heparin has been reported to be able to modulate TF expression, no information is still available on the pharmacological modulation of asTF in endothelial cells. Aim: To investigate the kinetic of asTF mRNA expression and the modulation of TF and asTF by unfractionated (UH) and low molecular weight heparin (LMWH). Methods: Endothelial cells (HUVEC) were incubated with heparins (0.001 10 UI/ml) and stimulated with LPS (1 mg/ml) or TNF-a (10 ng/ml). TF and asTF mRNA levels were quantified by Real Time PCR. TF pro-coagulant activity was also evaluated. Results: Stimulation of HUVEC with TNF-a resulted in a timedependent increase of both TF and asTF mRNA levels, with a peak of effect after 60 minutes stimulation for both transcripts. Treatment with UH or LMWH resulted in a concentration-dependent reduction of TF and asTF mRNA levels ( 40% and 30% respectively with the highest concentration). Both heparins were also able to significantly reduce a concentration dependent manner TNF-a and LPS-induced pro-coagulant activity. Conclusion: TNF-a and LPS stimulation of HUVEC induces a concomitantly upregulation of TF and asTF mRNA. The heparininduced down-regulation of asTF mRNA levels might further strengthen the antithrombotic effect of this drug.