Local coagulation activation has been shown to impact both primary tumor growth and metastasis in mice. It is well known that components of the blood clotting cascade such as tissue factor and... Show moreLocal coagulation activation has been shown to impact both primary tumor growth and metastasis in mice. It is well known that components of the blood clotting cascade such as tissue factor and thrombin play a role in tumor progression by activating cellular receptors and local formation of fibrin. However, whether venous thromboembolism (VTE) or a hypercoagulable state has a direct impact on cancer progression is unknown. Here we have combined an orthotopic murine breast cancer model, using female Nod-SCID mice, with siRNA-mediated silencing of antithrombin (siAT) leading to the induction of a systemic hypercoagulable state. We show that, compared to control siRNA-treated (not experiencing a hypercoagulable state) tumor-bearing mice, siAT treated tumor-bearing mice do not show enhanced tumor growth nor enhanced metastasis. We conclude that, in this murine model for hypercoagulability, induction of a hypercoagulable state does not contribute to breast cancer progression. Show less
Heestermans, M.; Salloum-Asfar, S.; Streef, T.; Laghmani, E.; Salvatori, D.; Luken, B.M.; ... ; Vlijmen, B.J.M. van 2019
Tissue factor, coagulation factor XII, platelets, and neutrophils are implicated as important players in the pathophysiology of (experimental) venous thrombosis (VT). Their role became evident in... Show moreTissue factor, coagulation factor XII, platelets, and neutrophils are implicated as important players in the pathophysiology of (experimental) venous thrombosis (VT). Their role became evident in mouse models in which surgical handlings were required to provoke VT. Combined inhibition of the natural anticoagulants antithrombin (Serpinc1) and protein C (Proc) using small interfering RNA without additional triggers also results in a venous thrombotic phenotype in mice, most notably with vessel occlusion in large veins of the head. VT is fatal but is fully rescued by thrombin inhibition. In the present study, we used this VT mouse model to investigate the involvement of tissue factor, coagulation factor XII, platelets, and neutrophils. Antibody-mediated inhibition of tissue factor reduced the clinical features of VT, the coagulopathy in the head, and fibrin deposition in the liver. In contrast, genetic deficiency in, and small interfering RNA-mediated depletion of, coagulation factor XII did not alter VT onset, severity, or thrombus morphology. Antibodymediated depletion of platelets fully abrogated coagulopathy in the head and liver fibrin deposition. Although neutrophils were abundant in thrombotic lesions, depletion of circulating Ly6G-positive neutrophils did not affect onset, severity, thrombus morphology, or liver fibrin deposition. In conclusion, VT after inhibition of antithrombin and protein C is dependent on the presence of tissue factor and platelets but not on coagulation factor XII and circulating neutrophils. This study shows that distinct procoagulant pathways operate in mouse VT, dependent on the triggering stimulus. Show less
Heestermans, M.; Jong, A. de; Tilburg, S. van; Reitsma, P.H.; Versteeg, H.H.; Spronk, H.M.; Vlijmen, B.J.M. van 2019
Silencing of anticoagulant protein C using RNA interference (siProc) evokes low incident but spontaneous atherothrombosis in the aortic root of apolipoprotein E-deficient (Apoe(-/-)) mice. The... Show more Silencing of anticoagulant protein C using RNA interference (siProc) evokes low incident but spontaneous atherothrombosis in the aortic root of apolipoprotein E-deficient (Apoe(-/-)) mice. The aims of the current study were (1) to analyze if plaque characteristics or circulating factors could be linked to atherothrombosis susceptibility, (2) to increase the incidence of atherothrombosis by transiently increasing blood pressure, and (3) to direct atherothrombosis to an additional predefined vascular site by applying a semi-constrictive collar around the carotid artery. siProc-driven spontaneous atherothrombosis in the aortic root of Apoe(-/-) mice was reproduced and occurred at an incidence of 23% (9 out of 39 mice), while the incidence of collar-induced atherothrombosis in the carotid artery was 2.6% (1 out of 39 mice). Treatment with phenylephrine, to transiently increase blood pressure, did not increase atherothrombosis in the aortic root of the Apoe(-/-) mice nor in the carotid arteries with collars. Plaques in the aortic root with an associated thrombus were lower in collagen and macrophage content, and mice with atherothrombosis had significantly more circulating platelets. Plasma protein C, white blood cell counts, total cholesterol, fibrinogen, serum amyloid A, and IL-6 were not different amongst siProc treated mice with or without thrombosis. Remarkably, our data revealed that thrombus formation preferably occurred on plaques in the right coronary sinus of the aortic root. In conclusion, there is a predilection of low protein C-induced spontaneous atherothrombosis in Apoe(-/-) mice for the right coronary sinus, a process that is associated with an increase in platelets and plaques lower in collagen and macrophage content.. Show less
Heestermans, M.; Ouweneel, A.B.; Hassan, J.; Kloosterman, M.; Reitsma, P.H.; Gijbels, M.J.J.; ... ; Eck, M. van 2018
During my research project we mainly focussed on studying the pathophysiology of venous and arterial thrombosis in mice. When we transiently lowered plasma protein levels of natural anticoagulants... Show moreDuring my research project we mainly focussed on studying the pathophysiology of venous and arterial thrombosis in mice. When we transiently lowered plasma protein levels of natural anticoagulants antithrombin and protein C using RNA interference, mice developed venous thrombosis in the head. In contrast to other mouse models for venous thrombosis where surgery is required for provoking the disease, mice injected with RNA interference against the mRNA of Serpinc1 and Proc (antithrombin and protein C, respectively) developed venous thrombosis without additional handlings. In this unique form of venous thrombosis, we studied the roles of platelets, neutrophils, and coagulation factor XII. These factors have been shown to be indispensable in experimental venous thrombosis in other mouse models, and they have been introduced as novel therapeutic targets. For the second part of my thesis we again used the RNA interference approach, to lower natural anticoagulation in atherosclerotic mice. When we lowered protein C in these mice, they developed atherothrombosis in the aortic root without any additional intervention. This unique form of atherothrombosis has been showed in multiple independent experiments, and we aimed to further characterize the process to learn more about prevention atherothrombosis in atherosclerotic mice and the role of protein C. Show less
Spronk, H.M.H.; Padro, T.; Siland, J.E.; Prochaska, J.H.; Winters, J.; Wal, A.C. van der; ... ; Cate, H. ten 2018
Murine atherosclerosis models do not spontaneously develop atherothrombotic complications. We investigated whether disruption of natural anticoagulation allows preexisting atherosclerotic plaques... Show moreMurine atherosclerosis models do not spontaneously develop atherothrombotic complications. We investigated whether disruption of natural anticoagulation allows preexisting atherosclerotic plaques to progress toward an atherothrombotic phenotype. Mice featured clots in the left atrium of the heart. Our findings indicate that small interfering RNA-mediated silencing of protein C in apolipoprotein E-deficient mice creates a condition that allows the occurrence of spontaneous atherothrombosis, albeit at a low incidence. Lowering natural anticoagulation in atherosclerosis models may help to discover factors that increase atherothrombotic complications. Show less
Ouweneel, A.B.; Heestermans, M.; Verwilligen, R.A.F.; Eck, M. van; Vlijmen, B.J.M. van 2016
Objectives: Studies on atherothrombosis, a major cause of cardiovascularevents, are hampered by the lack of animal models spontaneously developingatherothrombosis. Major underlying problems are the... Show moreObjectives: Studies on atherothrombosis, a major cause of cardiovascularevents, are hampered by the lack of animal models spontaneously developingatherothrombosis. Major underlying problems are the stability ofthe atherosclerotic plaques, combined with the strong plasma anticoagulantactivity. We hypothesized that inhibition of the anticoagulant systemby silencing of protein C (Proc) may cause murine atherosclerotic plaquesto become prone to atherothrombosis. Show less
Heestermans, M.; Cunha, M.L.R.; Reitsma, P.H.; Zeerleder, S.S.; Middeldorp, S.; Vlijmen, B.J.M. van 2016
