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CK2-dependent cytoskeletal regulation and molecular signaling of Neutrophil Extracellular Trap (NET) formation

Periodic Reporting for period 1 - COAGULANT (CK2-dependent cytoskeletal regulation and molecular signaling of Neutrophil Extracellular Trap (NET) formation)

Reporting period: 2018-09-01 to 2020-08-31

Cardiovascular diseases like myocardial infarction, stroke, and venous thromboembolism, as well as associated complications are still the leading cause of morbidity and mortality in the European Union, resulting in enormous health care costs. Although, it is well acknowledged that beside platelets the immune system plays a pivotal role in thrombus formation and cardiovascular conditions, the underlying molecular and signaling mechanisms are still poorly defined and cellular processes are not understood. Nevertheless, neutrophils were shown to significantly contribute to venous thrombosis and thromboembolism. Activated neutrophils are able to release decondensed chromatin in a process called Neutrophil Extracellular Trap formation (NETosis), which is well known to foster platelet activation and adhesion. Originally described as crucial part of the innate immune response against pathogens , NETosis is now understood as a main factor in venous thrombus formation and progression, which can be induced by local hypoxia, among other things. Just recently, NETosis was defined as a well-orchestrated process of cellular events, including cytoskeleton rearrangements, nuclear envelope breakdown and plasma membrane rupture .

For this reason, the overall objective of the underlying project is the investigation of new molecular targets in the NETosis process and their impact on venous thromboembolism. Molecules and signaling mechanisms that affect the nuclear envelope breakdown and plasma membrane rupture in neutrophils are in the focus of the research, since both processes are crucial hallmarks of NETosis. In the end, the project will help to identify new druggable targets and biomarkers associated with thrombo-inflammatory diseases.
So far, one promising molecular target was identified that significantly affects NETosis and NET-driven deep vein thrombosis. Using genetically modified mice, pharmacological inhibition of primary peripheral neutrophils and a wide variety of visualization techniques as well as molecular biology approaches, the identified molecular target could be shown to regulate nuclear envelope breakage and plasma membrane rupture of NETing neutrophils. Application of a stenosis-induced venous thrombus formation mouse model unraveled significantly impaired thrombus progression in mice lacking the identified molecule when compared with control mice. Remarkably, by means of immunofluorescence staining, it was also observed that crucial parts of the identified target are released in line with NETosis, thus opening the possibility to induce platelet activation and inflammation during thrombo-occlusive disorders. A corresponding manuscript was prepared and just recently submitted to a peer-reviewed journal.
The knowledge of cellular mechanism and signaling cascades underlying NETosis will contribute to the development of new cutting-edge and personalized therapies and prophylaxis regimen for thrombo-occlusive disorders and, in particular, venous thromboembolism. Therefore, the importance of the identified target will be evaluated in cardiovascular diseases in humans during the last year of the project. To this end, prospective and retrospective patient cohorts and plasma samples will be investigated using the identified molecular target and NETosis markers, such as cell-free DNA, as readouts. This approach will help to establish the relevance of the newly identified NETosis-associated molecule as a biomarker of cardiovascular diseases and, especially, of venous thromboembolism. This will be of major importance for the society, since the prevalence and mortality rate of venous thromboembolisms has not improved in the last 30 years and 50% of the patients suffering from deep vein thrombosis does not have a benefit from current treatments and prophylaxis approaches . In doing so, the project will be in agreement with the first societal challenge in the EU Horizon2020 program and will contribute to the improvement of life quality and outcome of cardiovascular diseases in the EU.