Lois W. Brüggemann

Experimental melanoma metastasis in lungs of mice with congenital coagulation disorders

Experimental animal studies as well as clinical trials have shown that interventions targeting the blood coagulation cascade inhibit cancer cell metastasis. These data support the hypothesis that congenital prothrombotic disorders, like factor V Leiden, facilitate metastasis whereas bleeding disorders, like haemophilia impede metastasis. To test this hypothesis, we subjected factor V Leiden and factor VIII deficient mice to a murine model of experimental lung metastasis. In this model, B16F10 murine melanoma cells are injected into the tail vein resulting in multiple lung metastases within 20 days. Both hemi‐ and homozygous factor VIII deficient mice were protected against lung metastasis compared to wild‐type littermate controls. In contrast, homozygous factor V Leiden mice developed more metastases than wild‐type littermates, whereas heterozygous carriers showed an intermediate number of pulmonary foci. Overall, these data show that a congenital susceptibility to either bleeding or thrombosis modifies the metastatic capacity of cancer cells in the bloodstream and suggest that procoagulant phenotypes are a risk factor for tumour metastasis.

Assessing the efficacy of the hedgehog pathway inhibitor vitamin D3 in a murine xenograft model for pancreatic cancer

The developmental Hedgehog (Hh) pathway has been shown to cause malignancies in the adult organism, specifically in the proximal gastrointestinal tract. Previous studies have used the Hh-inhibitory alkaloid cyclopamine to treat Hh-dependent tumor growth. The present study aimed to determine the efficacy and specificity of the recently discovered endogenous inhibitor of the Hh pathway, vitamin D3, on inhibition of pancreatic adenocarcinoma cell growth in vitro and in vivo. Vitamin D3 was found to inhibit cell growth specifically through inactivation of Smo and the downstream Hh pathway, rather than activation of the vitamin D3 receptor. However, in in vivo models vitamin D3 was not found to be effective against tumor cell growth.

Differential effects of anticoagulants on tumor development of mouse cancer cell lines B16, K1735 and CT26 in lung

Abstract Cancer progression is facilitated by blood coagulation. Anticoagulants, such as Hirudin and low molecular weight heparins (LMWHs), reduce metastasis mainly by inhibition of thrombin formation and L- and P-selectin-mediated cell-cell adhesion. It is unknown whether the effects are dependent on cancer cell type. The effects of anticoagulants on tumor development of K1735 and B16 melanoma cells and CT26 colon cancer cells were investigated in mouse lung. Tumor load was determined noninvasively each week up to day 21 in all experiments using bioluminescence imaging. Effects of anticoagulants on tumor development of the three cell lines were correlated with the fibrin/fibrinogen content in the tumors, expression of tissue factor (TF), protease activated receptor (PAR)-1 and -4 and CD24, a ligand of L- and P-selectins. Hirudin inhibited tumor development of B16 cells in lungs completely but did not affect tumor growth of K1735 and CT26 cells. Low molecular weight heparin did not have an effect on K1735 melanoma tumor growth either. TF and PAR-4 expression was similar in the three cell lines. PAR-1 and CD24 were hardly expressed by K1735, whereas CT26 cells expressed low levels and B16 high levels of PAR-1 and CD24. Fibrin content of the tumors was not affected by LMWH. It is concluded that effects of anticoagulants are dependent on cancer cell type and are correlated with their CD24 and PAR-1 expression.

Endogenous activated protein C is essential for immune-mediated cancer cell elimination from the circulation

Fibrinogen and platelets play an important role in cancer cell survival in the circulation by protecting cancer cells from the immune system. Moreover, endogenous activated protein C (APC) limits cancer cell extravasation due to sphingosine-1-phosphate receptor-1 (S(1)P(1)) and VE-cadherin-dependent vascular barrier enhancement. We aimed to study the relative contribution of these two mechanisms in secondary tumor formation in vivo. We show that fibrinogen depletion limits pulmonary tumor foci formation in an experimental metastasis model in C57Bl/6 mice but not in NOD-SCID mice lacking a functional immune system. Moreover, we show that in the absence of endogenous APC, fibrinogen depletion does not prevent cancer cell dissemination and secondary tumor formation in immune-competent mice. Overall, we thus show that endogenous APC is essential for immune-mediated cancer cell elimination.

Hyperglycemia accelerates arterial thrombus formation and attenuates the antithrombotic response to endotoxin in mice

Recent human studies reveal that hyperglycemia induces procoagulant and antifibrinolytic effects in blood that may contribute to a greater risk of arterial thrombosis, but the direct relationship between high blood glucose levels and thrombosis has not yet been investigated. We performed a number of experiments to clarify whether hyperglycemia was causally related to arterial thrombosis and whether the combined stimulus of hyperglycemia and inflammation would enhance the thrombotic effect. In a model of ferric-chloride-induced carotid artery thrombosis, hyperglycemia did not influence the time to occlusion in mice pretreated with streptozotocin, but the rate of thrombus formation was accelerated. This effect was associated with increased thrombin generation and could not be explained by changes in vessel-wall tissue factor activity. The prothrombotic effect of hyperglycemia was assessed in a separate experiment, showing that collagen/thrombin-induced platelet procoagulant activity was increased in hyperglycemic mice. The effect of inflammation was studied by injecting a low dose of endotoxin that caused a systemic inflammatory state after 24 h (increased plasma levels of tumor necrosis factor α, interleukin-6 and monocyte chemotactic protein 1 in diabetic and nondiabetic mice) associated with a mild delay in thrombus formation. This reduced rate of thrombus formation was attenuated by hyperglycemia. Together, these data establish a discrete but clear contribution of hyperglycemia in experimental arterial thrombosis.

Role of coagulation FVIII in septic peritonitis assessed in hemophilic mice

Summary.  Background: Inhibition of blood coagulation appears to be an important therapeutic strategy to improve the outcome in sepsis. However, the beneficial effect of anticoagulant treatment in sepsis is solely based on experimental data using inhibitors of the extrinsic coagulant pathway. The role of the intrinsic pathway of coagulation in the pathogenesis of sepsis has not been explored yet. Objective: In the current study, we contribute to determine the role of factor (F)VIII, the key player of the intrinsic coagulant pathway, on host defense against peritonitis. Method: Hemizygous FVIII‐deficient mice and their wild‐type littermates were challenged with 1 × 104 bacteria in a septic peritonitis model. Results: The intraperitoneal injection of Escherichia coli led to growth and dissemination of bacteria and provoked an inflammatory response as evident from elevated cytokine levels, increased cell influx into tissues, liver necrosis, and endothelialitis resulting in mortality. The FVIII‐deficient genotype slightly reduced bacterial outgrowth but had no effect on markers of inflammation and/or survival. In addition, FVIII‐deficient mice showed profound activation of coagulation, thereby improving the hemophilic phenotype of FVIII‐deficient mice. Conclusion: FVIII deficiency slightly modifies host defense in septic peritonitis in mice, but does not influence the final outcome of peritonitis. Therefore, we question the importance of the intrinsic coagulant pathway during sepsis.

Alternatively spliced tissue factor in mice: induction by Streptococcus pneumoniae

Alternatively spliced tissue factor (asTF) has recently been described in humans to be a soluble isoform of tissue factor (TF), which is present in blood. However, the existence of asTF is raising controversy in the field of haemostasis because active TF within the bloodstream would lead to massive intravascular thrombosis. Therefore the existence and biological role of asTF has been disputed ever since its discovery. The objective of this study was to prove or refute the significance of asTF in (patho) physiology. We investigated whether asTF is present in mice. To assess the potential involvement of asTF in (patho)physiology, we determined the effect of Streptococcus Pneumoniae infection and acute arterial thrombosis on asTF levels in mice. We show that mice also express a soluble TF variant lacking exon 5 and thus the transmembrane region. This murine asTF contains a novel C-terminus of 94 amino acids leading to a protein of 291 amino acids. The C-terminus of murine asTF shares strong homology with human asTF, is 54 amino acids longer and has no significant homology with any other protein in the NCBI database. Murine asTF is expressed in lung tissue, in which it is induced by S. Pneumoniae infection. Furthermore, murine asTF is present in plasma and can be found throughout arterial blood clots induced by FeCl3. The finding that mice produce asTF, which is induced by Streptococcus Pneumoniae and is omnipresent in blood clots, strongly suggest an important role for asTF in (patho)physiology.

Long-term thrombin inhibition promotes cancer cell extravasation in a mouse model of experimental metastasis

T . M . H . N IE R S , * L . W. BR ÜG GE MA NN , G. L . VAN SLU IS , * § R . D . L I U , H. H . VERSTEEG ,– H. R . B Ü LLER , C. J . F . VAN N OORDEN,§ P . H . RE ITSMA,– C. A . SPEK and D . J . R IC HEL* *Department of Medical Oncology, Academic Medical Centre, University of Amsterdam, Amsterdam; Centre for Experimental and Molecular Medicine, Academic Medical Centre, University of Amsterdam, Amsterdam; Department of Vascular Medicine, Academic Medical Centre, University of Amsterdam, Amsterdam; §Department of Cell Biology and Histology, Academic Medical Centre, University of Amsterdam, Amsterdam; and –Einthoven Laboratory for Experimental Vascular Research, Leiden University Medical Centre, Leiden, the Netherlands

Prophylactic plasma levels of the low molecular weight heparin nadroparin does not affect colon cancer tumor development in mouse liver

a Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands b Department of Medical Oncology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands c Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands d Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands