Alexander Steinhart

Deep vein thrombosis (DVT) is a major cause of cardiovascular death. The sequence of events that promote DVT remains obscure, largely as a result of the lack of an appropriate rodent model. We describe a novel mouse model of DVT which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of DVT in humans. We demonstrate by intravital two-photon and epifluorescence microscopy that blood monocytes and neutrophils crawling along and adhering to the venous endothelium provide the initiating stimulus for DVT development. Using conditional mutants and bone marrow chimeras, we show that intravascular activation of the extrinsic pathway of coagulation via tissue factor (TF) derived from myeloid leukocytes causes the extensive intraluminal fibrin formation characteristic of DVT. We demonstrate that thrombus-resident neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII) and by supporting its activation through the release of neutrophil extracellular traps (NETs). Correspondingly, neutropenia, genetic ablation of FXII, or disintegration of NETs each confers protection against DVT amplification. Platelets associate with innate immune cells via glycoprotein Ibα and contribute to DVT progression by promoting leukocyte recruitment and stimulating neutrophil-dependent coagulation. Hence, we identified a cross talk between monocytes, neutrophils, and platelets responsible for the initiation and amplification of DVT and for inducing its unique clinical features.

Fractalkine (CX3CL1, FKN) is expressed in the inflamed vascular wall and absence of FKN reduces atherogenesis. Whether FKN is expressed throughout all stages of atherosclerotic disease and whether it directly contributes to monocyte recruitment to atherosclerotic lesions is not known. We collected human atherosclerotic plaque material and blood samples from patients with carotid artery disease undergoing endarterectomy. Plaques were analyzed by immunohistochemistry and qPCR. We found that FKN is expressed at all stages of atherosclerotic lesion formation, and that the number of FKN-expressing cells positively correlates with the number of CX3CR1-positive cells in human carotid artery plaques. In the circulation, soluble FKN levels are significantly elevated in the presence of high-grade (sub-occlusive) stenosis. To determine the role of the FKN-CX3CR1 axis for monocyte adhesion in vivo we then performed intravital videofluorescence microscopy of the carotid artery in ApoE(-/-) mice. Notably, FKN-CX3CR1 interactions are critical for recruitment of circulating monocytes to the injured atherosclerotic vascular wall. Thus, this chemokine dyad could represent an attractive target for anti-atherosclerotic strategies.

Objective: Venous thromboembolism has a major medical impact and is the third leading cause of cardiovascular associated death. While the critical contribution of platelets to arterial thrombosis has been recognized, the mechanisms that trigger the development of deep vein thrombosis (DVT) are not yet fully understood and are believed to be mainly dependent on coagulation. Current in vivo models associated with endothelial disruption or complete stasis only incompletely reflect the pathophysiology of DVT in humans and hinder further understanding of this process. Here, we established a novel mouse model of DVT formation and assessed the dynamics of leukocyte (lc) and platelet (pt) recruitment in vivo. Methods: In our new model DVT was induced by reduction of blood flow in the inferior vena cava (IVC) (to 23.8% of baseline) in the absence of endothelial injury. Layered thrombi develop slowly over a prolonged period of time (>6-12hrs) and occlude the entire IVC 24-48hrs later mimicking human venous thrombi. We observed the dynamics of pt (GPIb-/-, GPIIb-/-), lc (CX3CR1-EGFP, LysM-EGFP), and microparticle recruitment in the IVC in vivo by intravital microscopy. Results: Using corresponding transgenic mice we show here that lc accumulation required endothelial P-selectin, and, besides monocytes, consisted mainly of neutrophils which contribute to DVT development by NET release. In addition to innate immune cells, pts are indispensable for the initiation of venous thrombosis. Further, using low-TF and hTF mice we demonstrate that blood derived tissue factor (TF), exposed on NETs and microparticles, creates a procoagulatory environment that is supported by pt adhesion and activation. The intrinsic pathway of coagulation is also involved in DVT formation, since a factor XIIa inhibitor markedly impaired DVT development. Conclusion: Together, venous thrombosis in our model is driven by a concerted interaction of pts, lcs, and the coagulation cascade. Pts bind to endothelial cells mediated by GPIbα and GPIIb-IIIa, recruited lcs, and triggered NET release thereby initiating DVT. A modulation can be attained in three different ways: by aiming on the coagulation cascade (heparins, factor inhibitors), by impairing pt function and/or by inhibiting lc activation.

Deep vein thrombosis (DVT) is a major cause of cardiovascular death. The sequence of events that promote DVT remains obscure, largely as a result of the lack of an appropriate rodent model. We describe a novel mouse model of DVT which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of DVT in humans. We demonstrate by intravital two-photon and epifluorescence microscopy that blood monocytes and neutrophils crawling along and adhering to the venous endothelium provide the initiating stimulus for DVT development. Using conditional mutants and bone marrow chimeras, we show that intravascular activation of the extrinsic pathway of coagulation via tissue factor (TF) derived from myeloid leukocytes causes the extensive intraluminal fibrin formation characteristic of DVT. We demonstrate that thrombus-resident neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII) and by supporting its activation through the release of neutrophil extracellular traps (NETs). Correspondingly, neutropenia, genetic ablation of FXII, or disintegration of NETs each confers protection against DVT amplification. Platelets associate with innate immune cells via glycoprotein Ibα and contribute to DVT progression by promoting leukocyte recruitment and stimulating neutrophil-dependent coagulation. Hence, we identified a cross talk between monocytes, neutrophils, and platelets responsible for the initiation and amplification of DVT and for inducing its unique clinical features.