Title Promoter Affiliations Abstract "Understanding how neutrophil extracellular traps (NETs) contribute to vulnerability of the ageing heart" "Kim Martinod" "Centre for Molecular and Vascular Biology, Experimental Cardiology, Cardiovascular Developmental Biology" "Ageing is a major risk factor for heart failure, which is a leading cause of mortality worldwide. Which factors and processes render the elderly heart more vulnerable to additional stressors, like hypertension, remains unclear. Growing evidence suggests that the hearts’ resident cells (cardiomyocytes and fibroblasts) are not the sole contributors to this increasing vulnerability. With ageing, the immune system undergoes a shift from adaptive to innate immunity. In addition, there is a constant low-grade level of inflammation in the ageing heart. Our interest lies in a key player in inflammatory and immune responses: the neutrophil. These innate immune cells play an important role not only in early phase responses to infections and tissue damage but also have long-term effects via their influence on other immune players that are recruited at later stages. One of their defense mechanisms is the formation of neutrophil extracellular traps (NETs), which involves releasing a web of DNA fibers lined with toxic proteins and histones. When this process becomes excessively activated it can be detrimental to the surrounding tissue and enhance the pro-inflammatory environment. Hereby, they contribute to downstream stress responses like scar formation, which promotes progression to heart failure. At this stage, the heart is no longer able to properly pump blood to the rest of the body. Absence of this NET response in genetically modified preclinical models reduces signs of cardiac ageing and preserves heart function in aged animals. We will study these older animals along with their younger counterparts and identify differences in expression, functionality and physiology of the different cell types involved in cardiac aging. By comparing neutrophil responses to cardiac stressors in aged vs. young preclinical models, we aim to understand how this protective phenotype is established and how neutrophils and NETs contribute to vulnerability of the ageing heart. We will also identify other mediators of the immune system that are involved in the stress response and could accelerate or slow down heart failure. New insights in these mechanisms contributing to cardiac vulnerability could guide future developments of therapeutic strategies to expand health span and ameliorate quality of life of the elderly." "Neutrophil extracellular traps (NETs) in ischemic stroke: from basic insights to potential therapies" "Simon De Meyer" "Cardiovascular Sciences, Kulak Kortrijk Campus" "Stroke is one of the leading causes of death and disability worldwide. Strikingly, the paramount medical relevance of ischemic stroke is in strong contrast to the limited treatment options. Indeed, only one pharmacological strategy is currently approved for acute stroke treatment: rapid thrombolysis of the occluding thrombus using tissue plasminogen activator (t-PA). However, use of t-PA has many serious limitations, including risk of bleeding, narrow therapeutic time window and neurotoxic effects. Although timely recanalization of the occluded cerebral artery is fundamental to salvage threatened ischemic tissue, reperfusion of the ischemic territory can also seriously exacerbate tissue damage by reperfusion injury, further worsening clinical outcome.The development of novel therapies is hampered by our incomplete understanding of the complex cellular and molecular interactions underlying stroke pathology. The “thrombo-inflammatory” nature of stroke, involving a complex interplay between both thrombotic and inflammatory processes, has been widely accepted. An intriguing new link between thrombosis and inflammation has just recently been discovered: neutrophil extracellular traps or NETs. A growing body of evidence reveals that NETs also form in human thrombosis and that NET biomarkers in plasma reflect disease activity. In this project, our aim is to investigate the involvement of NETs in ischemic stroke as this could open up novel treatments avenues in stroke management." "Towards diagnostic use of Neutrophil Extracellular Trap (NET) measurements in patient samples by pairing novel biomarker measurements with established microscopy readouts" "Kim Martinod" "Centre for Molecular and Vascular Biology, Semmelweis Egyetem" "Without a doubt, neutrophil extracellular traps (NETs) have led to a paradigm shift in the field of thrombosis and hemostasis, and broadly in other disease pathologies. However, in terms of measuring NETs in blood samples, the field suffers from a lack of standardization of methodology, with methods using unspecific readouts often interpreted as NETs (i.e. DNA which could come from other cell death). Our aim is thus to provide scientific backing for the use of certain assays to detect NET biomarkers as appropriate readouts for the complex biological process going on in human disease. We will do this by pairing established microscopy methods and with biochemical assays used to detect NET fragments as biomarkers. A direct comparison of assays measuring complexes of NET components will be compared to individual component measurements. This is a first step toward implementation of NETs measurements for diagnostic purposes, which is currently not possible. With a better understanding of how to accurately measure NETs in blood samples by pairing what we see by microscopy in research settings with biochemical measurements that can be applied in clinical laboratory settings, we will be able to start to bridge the gap from the bench to the bedside, where NET measurements can provide valuable insight into disease progression in a range of cardiovascular and inflammatory diseases." "Standardization of neutrophil extracellular trap (NET) measurements in patient samples." "Kim Martinod" "Centre for Molecular and Vascular Biology" "White blood cells are essential for fighting infection using specific killing mechanisms. However, when these processes get activated in the absence of infection, this can cause disease. One of the ways that white blood cells try to fight infection is by sending out neutrophil extracellular traps (NETs), ejecting their DNA lined with proteins that can kill bacteria. These same proteins also damage healthy blood vessels and cause them to become activated, which binds platelets and can lead to blood clots. Activated platelets are also one way that white blood cells can be stimulated to make NETs, and therefore this is a vicious cycle. However, in terms of measuring these NETs in blood samples, the field suffers from a lack of standardization of methodology, with some methods using unspecific readouts interpreted as NETs (for example, DNA which could come from other cell death). The aim of this project is begin a larger standardization project measurements of NETs in the field of thrombosis and haemostasis, which we hope will also be adopted by other fields. This also is a first step toward implementation of NETs measurements for diagnostic purposes, which is currently not possible." "Neutrophil extracellular traps in cardiac fibrosis - Peptidylarginine deiminase 4 and neutrophil extracellular traps in chronic inflammation and cardiac fibrosis" "Kim Martinod" "Centre for Molecular and Vascular Biology" "Heart failure is a broad term encompassing a multitude of clinical symptoms and signs. A finer distinction is currently being made, subdividing this one term into different subtypes based on the remaining systolic function of the patient. On a global scale, HF is estimated to affect approximately 64 million people, making it a pandemic problem. Although the pathology is long recognized, and the subject of extensive research over the last decades, therapies are still limited, mainly focusing on treating symptoms, rather than the underlying pathophysiology. This means that, although morbidity is reduced, overall mortality due to HF remains high. Treatment of HF remains troublesome due to the complex underlying molecular mechanisms of the disease, which includes cardiac fibrosis. This can result in remodeling of the heart tissue, and more specifically of the extracellular matrix (ECM), due to an increase in the deposition of mainly collagen, leading to stiffness of myocardium. This eventually results in a stiffening of the heart muscle. This cardiac remodeling starts off as an essential and beneficial process during tissue injury. However, when injury to the tissue, in the case of HF the heart, becomes repetitive or prolonged, this beneficial process of tissue healing evolves into excessive deposition of ECM proteins, thereby altering the architecture of the underlying tissue, resulting in a diminishment of function finally leading to organ failure, which in case of the heart tissue is equivalent to heart failure development.  One mechanism leading to tissue fibrosis is chronic inflammation. However, the exact underlying cellular and molecular mechanisms driving this process are insufficiently understood to have translated into any current therapeutic options. During chronic inflammation, cells of the immune system are in a constant state of preparedness. In neutrophils, this means there is a low threshold for the formation of neutrophil extracellular traps (NETs) due to activation of the enzyme peptidylarginine deiminase 4 (PAD4). These NET structures have a role in preventing pathogen dissemination throughout the body upon infection of the host. However, once released in an unregulated manner, such as in sterile chronic inflammation, NETs have been shown to be both prothrombotic and proinflammatory.  In addition, NETs are known to cause uncontrolled fibrosis in the lung during chronic inflammation, and systemic deletion of PAD4 prevents spontaneous cardiac fibrosis with increasing age. Therefore, we hypothesize that neutrophils, via NETs, contribute centrally to chronic inflammation-induced cardiac remodeling. To understand the impact of activated neutrophils and NETs on the heart, a genetic mouse line of PAD4 deletion under a neutrophil-selective promoter (Ne-PAD4-/-) was generated and confirmed to have a deficiency in releasing NETs. This mouse strain, together with appropriate controls, was then exposed to different settings of chronic inflammation, after which the effect on the heart muscle was evaluated on both a functional and structural level.  During the process of natural aging, there is a shift in the composition of the immune system, resulting in a decline in the adaptive immune system while the innate immune system gains importance. In combination with this shift, natural aging is accompanied by a chronic low-grade form of inflammation, which is referred to as ‘inflammaging.’ To understand the effect of this physiological process of inflammaging, and the contribution of neutrophils, our NET-deficient mice were aged for a period of two years, coinciding with 70 years in humans. Following this period of natural aging, cardiac function was evaluated through transthoracic echocardiography, while histology of the heart tissue was used to understand changes in cardiac structure following a prolonged period of chronic inflammation. After this period of natural aging, mice deficient in NET formation maintained cardiac function (both systolic and diastolic) comparable to young and healthy control mice. After further structural evaluation of the heart muscle, these mice were found to have both a decreased collagen expression level as well as decreased levels of both interstitial and perivascular collagen deposition in the heart as compared to wild-type controls. In addition, following two years of aging, cardiac hypertrophy was absent in Ne-PAD4-/- while wild-type controls showed extensive hypertrophy due to the increase in age. This cardiac remodeling and functional deterioration in wild-type but not Ne-PAD4-/- was later attributed to an increased level of inflammation in wild-type mice compared to Ne-PAD4-/- mice, evaluated through blood composition and evaluation of circulating inflammatory markers.   In order to confirm these findings of NET dependent cardiac remodeling following prolonged periods of chronic inflammation, two additional mouse models of chronic inflammation were adopted that mimick human comorbidities or cardiovascular diseases commonly associated with heart failure. In a first model, mice were exposed to prolonged metabolic stress in the form of high fat intake obesity development and progression. As it is well established that suffering from obesity results in an increased activation of the immune system, similar findings were expected. Interestingly, Ne-PAD4-/- mice did not gain body wheight in a similar fashion compared to wild-type mice, despite food intake being comparable between the groups over the experimental period. In addition, the prolonged period of high fat intake (10 weeks) and subsequent obesity development, resulted in a decrease in cardiac diastolic function in wild-type mice capable of forming NETs, while in NET-deficient animals' cardiac deterioration was absent. These observations again highlight the importance of neutrophils and NETs beyond their role in the immune system. In a final model, chronic systemic inflammation was established through a model of deep vein thrombosis through inferior vena cava ligation (IVC). The mechanical induction of thrombus formation through IVC stenosis resulted in increased levels of NET formation by circulating neutrophils. Following this observation, cardiac function and remodeling were again investigated. In concordance with the previous observations following chronic inflammation, wild-type mice again showed to be prone to the development of both functional and structural impairments with a decrease in systolic function and an increase in cardiac hypertrophy, two weeks after the establishment of a thrombus through IVC ligation.   The observed absence in cardiac damage in Ne-PAD4-/- mice following chronic periods of inflammation seems to be in part due to a reduction in neutrophil movement and recruitment following activation. A possible explanation for this is the contribution of PAD4 to the formation of the NLRP3 inflammasome. This multiprotein complex is assembled following neutrophil activation and requires the presence of PAD4. We established that the NLRP3 inflammasome is essential in directional movement of neutrophils towards an in vitro gradient of chemoattractant, and thus might be essential in tissue recruitment of neutrophils following injury or damage. This mechanistic insight into neutrophil function may have future implications on research into the impact of neutrophils in cardiovascular diseases.   In conclusion, this thesis work elucidates the detrimental effects of neutrophils, especially regarding the heart muscle, following chronic inflammation and proposes PAD4 as a crucial enzyme responsible for the acquired damage, either through NET formation or neutrophil motility. Therefore, this further supports that PAD4 is a clear therapeutic target to pursue in future research and development.  " "Neutrophil extracellular traps in cardiac fibrosis and heart failure" "Kim Martinod" "Centre for Molecular and Vascular Biology" "Neutrophil extracellular traps (NETs) have led to a paradigm shift in studying thrombotic and inflammatory disease. NETs promote thrombus formation by providing a scaffold for platelet, red blood cell, and coagulation factor binding. We and others have recently described the contribution of NETs and the early NET inducer peptidylarginine deiminase 4 (PAD4) to cardiac fibrosis development during aging in mice. Strikingly, aged PAD4-/- mice maintained healthy heart function (systolic and diastolic) comparable to young mice. In an experimental model of pressure-overload injury, both PAD4-deficiency and DNase treatment, which degrades NETs, similarly protected mice from aberrant collagen deposition and decline in cardiac output. In patients, this type of decline in cardiac function is linked to progression to heart failure with poor prognosis, often due to an increase in cardiac fibrotic remodeling with unknown etiology. It is interesting to study NET formation in the context of heart failure development. Our research lines will be to study sterile cardiac injury leading to heart failure with a focus on NETs and protein citrullination. We should obtain valuable insight into the role of neutrophil NETs or PAD4 in the pathogenesis of heart failure in patients, which could be applied to future therapeutic approaches in the clinic." "Investigating the impact of neutrophil extracellular traps and peptidylarginine deiminase 4 in Gram-positive sepsis" "Kim Martinod" "Centre for Molecular and Vascular Biology" "Sepsis is a significant health care problem, with mortality rates remaining at 30% despite years of research and implementation of new intervention therapies. The pathologies of sepsis result not only from an infection, but also from the hyperinflammatory host response. Most research, particularly in animal models, is currently on polymicrobial sepsis or Gram-negative bacterial products, which does not necessarily match the clinical cases of Gram-positive sepsis, including Staphylococcus aureus. S. aureus is known to be causative in infectious pathologies, including skin abscesses, endocarditis, and sepsis. S. aureus is a potent inducer of neutrophil extracellular traps (NETs), or protein-containing DNA strands released from white blood cells. These NETs form during S. aureus systemic infections and likely promote an exacerbated immune response and vascular occlusion. With this project, we aim to understand the virulence factors on S. aureus which drive NET formation, the role of the blood protein VWF in promoting NET formation, and the mechanistic role for the enzyme peptidylarginine deiminase 4 (PAD4) in promoting disease pathology. PAD4, also released on NETs, can modify extracellular proteins such as those found in plasma, and therefore could alter the disease profile in sepsis. In this project, we will determine if PAD4 released during sepsis is originating from neutrophils in mice and in patients. With this we will aim to understand the role of NETs in sepsis pathology." "Investigating the interplay between VWF, platelets, and neutrophil extracellular traps inpathologies involving thrombosis of the microvasculature" "Simon De Meyer" "Cardiovascular Sciences, Kulak Kortrijk Campus, Centre for Molecular and Vascular Biology" "Thrombosis is a complex process involving activation of endothelial cells, and their release of Weibel-Palade body contents such as von Willebrand factor (VWF) and P-selectin. This in turn recruits platelets which form aggregates, and leukocytes such as neutrophils. The recent discovery of neutrophil extracellular traps (NETs) described a novel antimicrobial function of neutrophils. NETs are extracellular chromatin strands containing microbicidal proteins. The release of histones, serine proteases, and myeloperoxidase concentrated on DNA fibers into the extracellular space can contribute to many pathologies. NETs can bind platelets and red blood cells and therefore also contribute to thrombosis. Activated platelets can in turn stimulate neutrophils to make NETs, creating a vicious thrombo-inflammatory cycle.Our first objective is to study VWF involvement in the induction of NETosis. We will elucidate the role of VWF as an important mediator of NET formation using VWF mutants. Innovative live-cell imaging and flow cytometry will be used to unravel the kinetics of VWF/platelet-mediated NET formation. Our second objective is to assess the physiological relevance of (inhibiting) platelet/VWF involvement in NETs-related pathology. We will investigate the role of VWF/NET interactions in three critical clinical settings where microvascular thrombosis contributes to multiorgan failure and/or mortality: sudden inflammatory response syndrome (SIRS), thrombotic thrombocytopenic purpura (TTP), and severe malaria. Mouse models of disease will be used and combined with several strategies for inhibition of VWF/platelet/NETs interactions to assess their effect on disease progression.In summary, with this research project we aim to better understand how platelets and VWF can drive NET formation in thrombo-inflammation, as this may also lead to more targeted therapeutic approaches in diseases which currently have limited treatment options." "Investigating the interplay between von Willebrand factor, platelets, and neutrophil extracellular traps in pathologies involving thrombosis of the microvasculature" "Kim Martinod" "Centre for Molecular and Vascular Biology" "White blood cells are essential for fighting infection using specific killing mechanisms. However, when these processes get activated in the absence of infection, this can cause disease. Similarly, von Willebrand factor (VWF), which serves as a sort of glue during blood clotting by sticking platelets (small blood cells) to damaged blood vessels, can also lead to disease when it gets released without injury. One of the ways that white blood cells try to fight infection is by sending out neutrophil extracellular traps (NETs), or ejecting their DNA lined with proteins that can kill bacteria. These same proteins also damage healthy blood vessels and cause them to release more VWF, which binds platelets and can lead to small blood clots. Activated platelets are one way that white blood cells can be stimulated to make NETs, and therefore this is a vicious cycle. We aim to better understand how VWF and platelets can trigger this release of NETs from white blood cells and to see if this occurs in diseases where small blood clots form in important organs leading to organ failure and sometimes death. By inhibiting these interactions in mouse experimental models, we hope to see a protective effect in disease states. We hope to gain insight into new treatments for these deadly diseases where small blood clots lead to organ failure." "Investigating the role of neutrophil extracellular traps in the failing heart" "Kim Martinod" "Centre for Molecular and Vascular Biology, Cardiology" "Neutrophils can release their nuclear contents lined with granule proteins, creating fibrous DNA nets with antimicrobial properties, called neutrophil extracellular traps (NETs). NETs also promote thrombus formation by providing a scaffold for platelet and clotting factor binding. The release of NETs thus shifts from a protective, immune defense mechanism, to a process with pathological consequences. The early NET inducer peptidylarginine deiminase 4 (PAD4) contributes to cardiac fibrosis development in mice. PAD4-deficiency and NET degradation similarly protected from aberrant collagen deposition and decline in cardiac output after injury. In humans, this type of decline in function is linked to progression to heart failure (HF) with poor prognosis, often due to an increase in cardiac fibrotic remodeling with an unknown cause. HF occurs when the heart cannot properly pump blood to the rest of the body. When heart muscle is damaged and tries to repair itself, widespread scar tissue (fibrosis) is left behind. The goal of this project to investigate how NETs contribute to this process. We will: 1) Delineate the kinetics of NET formation after cardiac injury; 2) Follow the downstream consequences of modulating NET formation and 3) Investigate NETs in patients at high risk of developing HF. With this project we aim to better elucidate HF pathogenesis and to provide insight into future diagnostic use of NETs in the context of HF and thrombosis."