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Platelets: at the nexus of brain aneurysm

Periodic Reporting for period 1 - PANDORA (Platelets: at the nexus of brain aneurysm)

Reporting period: 2016-12-01 to 2018-11-30

In the European Union, 15 million people have an unruptured intracranial aneurysm (IA) that may rupture one day and lead to subarachnoid hemorrhage (SAH). The IA rupture event is ominous and lingers as a clinical quandary. No safe and effective non-invasive therapies have, as of yet, been identified and implemented in clinical practice mainly because of a lack of knowledge of the underlying mechanisms. Preventive treatment of IA before rupture is therefore needed. Surgical clipping and endovascular therapy are invasive procedures with potentially serious complications Increasing evidence points to inflammation as one of the leading factors in the pathogenesis of IA. Intrasaccular clot formation is a common feature of IA occurring unruptured and ruptured IA. In addition to forming clots, activated platelets support leukocyte recruitment. Interestingly, platelets also prevent local hemorrhage in inflammatory situations independently of their ability to form a platelet plug. We hypothesize that the role of platelet may evolve throughout the development of IA: initially playing a protective role of in the maintenance of vascular integrity in response to inflammation and contributing later to intrasaccular thrombus formation. What are the platelet signaling pathways and responses involved and to what extent do they contribute to the disease and the rupture event?
The contribution of platelets to the maintenance of vascular integrity in inflammation does not depend on the platelet’s ability to form a hemostatic plug. At this point, very little is known about how platelets protect the inflamed vasculature. Recent studies, including mine, have identified a critical role for ITAM platelet signaling in the maintenance of vascular integrity at sites of inflammation. Surprisingly, the GPCR signaling, playing an instrumental role in platelet plug formation, is not required in supporting vascular integrity at sites of inflammation. Therefore, depending on the signaling pathway, platelets may release vasoactive factors, up to now not-defined, can prevent hemorrhage by strengthening EC barrier function or by dampening the inflammatory response. To answer that question, we performed in vitro studies in which platelets were incubated with neutrophils stimulated or not by TNFα 10ng/ml for 4hours. Peroxydase activity and reactive oxygen species were measured by fluorogenic substrates. We observed that although platelets are known to recruit leukocyte at sites of inflammation, they can also dampen neutrophil activation. We are currently performing experiments to further understand the exact platelet mechanisms involved. For that purpose, we will use platelets treated with different inhibitors (aspirin, P2Y12 inhibitor, anti GPVI…) and analyze neutrophil neutrophil response.

In parallel to the in vitro studies, we set up the in vivo model of intracranial aneurysm. For this purpose, I collaborated with Cyrille Orset at INSERM 919, University of Caen, France. I went to learn the model and implemented in my lab. It’s a finicky preclinical in-vivo animal model described by Nuki et al, analog to the clinical manifestation of the disease process. We implanted mice angiotensin pump to induce hypertension and then injected elastase into the cerebrospinal fluid are combined to induce intracranial aneurysm formation and rupture in mice monitored by MRI surgery. This model requires a lot of training and high skill level in microsurgery. Now, I am pretty confident with this model to further delineate the platelet mechanisms. Aside the learning curve of this model, I observed by immunohistology a thrombus in the aneurysmal sac highly enriched in platelets and leukocytes suggesting that platelets can play an instrumental role in this disease. I presented these results at seminars and open doors days in our laboratory. I also communicate this data through the class I am teaching at the University of Paris Diderot and at meetings (International Vascular Biology Meeting in Boston, European Congress on Hemostasis and Thrombosis in Den Hague and in Marseille 2018).
Understanding platelet mechanisms that contribute to the development of an intracranial aneurysm will be a major step forward in patient care in regard to alleviate the symptoms and improve the quality of life. The standard care of IA is the use of endovascular devices (flow diverter, coil) which is a major invasive procedure, often associated with a significant risk of disability. Deciphering platelet mechanisms involved during the development of IA will provide suitable targets and could potentially represent an alternative approach to the current treatment. First, we expect that this project will allow us to (i) identify platelet responses that can regulate leakage of the blood-brain barrier, (ii) decipher platelet signaling that can dampen leukocyte activity, (iii) platelet mechanisms that can regulate the blood-brain barrier integrity against leukocyte attacks. Second, with our in vivo model, we expect (i) to define platelet mechanisms involved in IA formation and rupture, (ii) to test current antiplatelet therapy and identify new targets in the IA model, (iii) to define an efficient therapeutic window to intervene on platelet activation during IA.
The ultimate goal of biomedical research is to develop novel and better therapeutic approaches for the treatment of human diseases. Importantly, cost of vascular brain diseases is considerably increasing as the population aged. This situation urges the need for new therapeutic strategy as the management of these patients is resource-intensive and has significant implications on public health, health budgets, and the education of future health professionals. Taking this into account, the overall aim of the project is to ease the social burden of an intracranial aneurysm by providing a framework for developing novel strategies and identify new therapeutic targets. We are certain that the potential clinical applications of the knowledge generated within this project are in line with the continuous effort of the European scientific community to be at the forefront of translational medicine.