Marfan syndrome (MFS) is a connective tissue disorder associated with autosomal dominant inheritance. MFS is caused by mutations in the FBN1 gene (15q21), responsible for fibrillin-1 protein deficiency or dysfunction. MFS is a multisystem disorder with manifestations in the skeletal, cardiovascular, pulmonary, and ocular systems and in skin, and dura. Cardiovascular disorders and more specifically aortic dissections are the most life-threatening clinical manifestations of MFS, mostly preceded by aortic root dilation occurring in approximately 80% of the MFS patients. Therapeutic options for MFS aortopathy are limited including ß-blockers and type 1 angiotensin receptors antagonists whose effectiveness is not optimal. Replacement of the ascending aorta is the most important manner to prevent aortic dissection and rupture but surgery remains associated with morbidity, mortality and need for re-operation. Therefore, more efforts have to be deployed towards the development of therapeutic approaches targeting pathophysiological pathways involved in MFS aortopathy progression and complications. The pathophysiology of MFS is complex combining fibrillin deficiency, elastin layer degradation, smooth muscle cell dysfunction and death. There are accumulating evidence that immune-inflammatory responses are locally impaired in MFS patients with the accumulation of vascular tissue macrophages and local release of pro-inflammatory cytokines and chemokines. Based on promising preliminary results in murine models of MFS, we hypothesized that TREM-2 a receptor expressed by vascular macrophages orchestrates local inflammatory responses and protects against both aortic aneurysm progression and complications. In this ambitious project combining experiments in murine models and analysis of human samples, we aim to explore the role of TREM-2 in the pathophysiology of MFS and to test a therapeutic approach using agonistic anti-TREM-2 antibody. We will use different mouse models to assess the effects of global TREM-2 deficiency or specific TREM-2 deficiency in macrophage subsets (monocyte-derived or resident) on aortic pathology. We will study the consequences of TREM-2 deficiency on matrix remodeling, the local inflammatory response and ultimately on aortic dilatation and its complications. Therapeutic approaches using monoclonal antibodies to stimulate TREM-2 will be evaluated at early and late stages of the pathology in a murine model of Marfan syndrome. Finally, the clinical relevance of our data will be assessed on human aortic tissue from Marfan patients.

Since the 1990’s, recombinant tissue plasminogen activator (rt-PA) has been the most effective and sole clinically approved drug to induce vessel recanalization in acute thrombotic events such as ischemic stroke. However, due to its short half-life, high doses of rt-PA have to be injected which generate a cascade of events in the circulation leading to serious side effects such as bleeding complications, modulation of the permeability of the blood-brain-barrier and intracranial hemorrhages. The number of patients that could benefit from rt-PA based treatment is significantly limited due to all of these safety-related restrictions. It is estimated that less than 5% of the patients receive a rt-PA treatment and, out of them, 60% either suffer permanent disabilities or die in the case of acute ischemic strokes. So, there is still a dire need for safe and noninvasive treatments. The development of new formulation of thrombolytics gains more and more interest to open new perspectives for clinical thrombolytic therapy with the aim to reduce the dose and, therefore, hemorrhagic side effects. In addition to the development of new fibrinolytic agents, a promising strategy based on Nanomedicine is propose here. The aim of FightClot is to develop innovative medical devices for the visualization and the targeted treatment of thrombotic diseases and particularly for stroke. Partner 1 (INSERM U1148) has previously demonstrated that fucoidan, an abundant and cost-effective marine polysaccharide with sulfated chains, exhibits a high affinity for P-selectin in vitro and in vivo which is overexpressed at the surface of endothelial cells and activated platelets during thrombotic diseases. The Laboratory developed micro- and nano-carriers functionalized with fucoidan and loaded with rt-PA to achieved the molecular diagnosis and targeted therapy in vitro and in vivo of cardiovascular diseases. To promote the targeted treatment of thrombotic diseases, Partner 1 will synthesize, thanks to these preliminary data, echogenic functionalized polymer microbubbles loaded with the new fibrinolytic drug produced by Partner 2 and able to be visualized and destroyed (sonoporation) by the ultrafast ultrasound sequences developed by Partner 3 (UMR 7371). To answer the safety-related restrictions of the clinical use of rt-PA, Partner 2 (INSERM U1237) will develop new fibrinolytic drugs with the production of an original double mutant of human tissue plasminogen activators displaying a fibrinolytic activity similar to that of the wilt type t-PA but without neurotoxicity which will be loaded onto microbubbles. To enhance thrombolysis Partner 3, which has a huge experience in high resolved, functional and vascular imaging using ultrafast ultrasound apparatus, along with ultrasonic drug-delivery, has developed two techniques to highlight microbubbles i) flowing microbubbles can be separated from tissue with spatio-temporal filters, a technique that is used for ultrasound localization microscopy of the brain, ii) stationary or targeted microbubbles can be separated using ultrafast radial modulation imaging. Finally, to validate the thrombolysis efficacy of the echogenic functionalized microbubbles, Partner 2 will use two preclinical stroke models in mice with in situ clot formation which mimic the main etiologies of the ischemic stroke in clinic. One done by local application of FeCl3 to induce platelets rich and tPA-resistant clots, and the other by local infusion of thrombin to generate fibrin rich and tPA–sensitive clots. FightClot is a disruptive project on ultrasound molecular diagnosis and targeted therapy of thrombotic diseases to answer the unmet medical need of acute clinical events treatment. By starting with preliminary results achieved by the Partners, FightClot project should succeed in to reach the endpoints proposed at ANR.

Hemophilia A and B are inherited bleeding disorders that result in impaired thrombin generation. Correction of bleeding in hemophilia patients is best achieved by the therapeutic administration of factor VIII (FVIII) or factor IX (FIX), the respective missing coagulation factors. Replacement therapy is however complicated by the short half-lives and immunogenicity of the therapeutic molecules. Alternatives to replacement therapy include bypassing agents such as activated factor VII or Emicizumab, a bispecific monoclonal antibody (mAb), or molecules that neutralize natural anticoagulant proteins such as TFPI, antithrombin or activated protein C. These molecules are delivered systemically and do not accumulate at the site bleeding. Hence, they do not fully compensate for the missing coagulation factor and may trigger potentially life-threatening thrombotic events. AAV-based gene therapy has brought encouraging results for hemophilia but is restricted to patients who have not developed anti-drug antibodies (ADA) to the therapeutic coagulation factor or to AAV, and may be associated with liver toxicity, at least in HA patients. There is thus a need for novel therapeutic molecules that specifically reinforce thrombin generation at the very bleeding site and only at the time of vessel injury. Our previous work demonstrates the importance of the natural anticoagulant protease nexin-1 (PN-1, or serpinE2) as a major but somewhat ignored regulator of thrombin. We demonstrated that blocking PN-1 corrects the hemophilia phenotype. PN-1 is expressed ubiquitously including by platelets. Our working hypothesis proposes that the targeted neutralization of platelet-released PN-1 should reinforce thrombin generation at the very bleeding site and only when it is required. To validate our hypothesis, we will develop an array of bispecific chimeric molecules made of different nanobodies (VHH), which we refer to as Nanochimeras: a pole of the Nanochimeras will neutralize PN-1 activity; the second pole will bind GPVI, a transmembrane glycoprotein protein specifically expressed by platelets and megakaryocytes. The Nanochimeras will thus concentrate PN-1-neutralizing VHHs at the platelet surface. Some of the Nanochimeras will be fused to the Fc portion of the human (hu) IgG1 to confer increased in vivo half-life, and potentially longer therapeutic efficacy. Our preliminary data include 3 VHHs that neutralize hu and mouse PN-1 to different extents, 6 VHHs that bind two different epitopes on huGPVI with nanomolar affinities and do not activate platelets or prevent collagen-induced platelet activation, and the proof that huPN-1 is functional in mouse plasma. We also started backcrossing knock-in huGPVI transgenic (Tg) mice on FVIII-deficient (HA) mice. Using these molecules and the technologies already in place in the two partner laboratories, we will pursue the following objectives: i) generate VHHs binding huGPVI with subnanomolar affinities that do not interfere with platelet functions, and VHHs with optimized PN-1 neutralizing activity, ii) generate and functionally validate recombinant bispecific Nanochimeras, iii) generate and validate a novel preclinical model of huPN-1 Tg huGPVI Tg HA mice, and iii) determine the in vivo half-life of the Nanochimeras, confirm their therapeutic potential in correcting thrombin generation, delineate the extent of thrombus formation and predict their immunogenicity. While the proof of concept will be obtained in a preclinical model of HA, our Nanochimeras will be suitable to patients with HA or HB, irrespective of whether they have developed ADA to FVIII or FIX, patients with other bleeding disorders characterized by insufficient thrombin generation, as well as HA patients who experience breakthrough bleeds while treated with Emicizumab.

Pain is one of the most distressing symptoms during the early postoperative period. Pain also occurs when rigid sealants are employed to stick tissues or close wounds as they hurt fragile tissues. To the best of our knowledge there is yet no sealant able to connect tissues in a soft manner. In this context, our project aims to develop original “soft glues” to efficiently connect tissues without mechanical constraints. SoftGlue will be elaborated in a one-step, easy to be scaled-up method, in water, at room temperature and without the need of organic solvents. In vitro studies will be carefully designed to minimize animal experiments, better understand the mechanisms of action of SoftGlue and optimize the formulations. First, in vitro sealing properties of SoftGlue will be assessed and compared to commercial products. Mechanical properties will be characterized using a set of standard procedures. In addition, we will develop an original method to study the bioadhesive properties, using an optical device to visualize SoftGlue in tissues under mechanical stresses (effect of thermo - mechanical solicitations) mimicking in vivo bonding wounds, blood flow, etc. After in vitro evaluations, selected few (3-4) SoftGlue will be evaluated in vivo. Tissue regeneration will be evaluated and neovessels formation will be investigated. Cell proliferation and migration will be assessed. Finally, a detailed diagram of the possible in vitro and in vivo toxic and genotoxic effects will be established. This project is a real opportunity to develop a long-term collaboration between complementary teams and to get this consortium persisting beyond scope of the project. SoftGlue is an ambitious interdisciplinary project dealing with an important societal need.

Metallic stents are widely used in endovascular treatment of coronary stenosis (balloon-expandable stents) and cerebral aneurysms (self-expandable flow diverters). In spite of the different design, both types of device are hampered by potentially life-threatening complications linked to the reaction of the blood elements and of the arterial wall in contact with the foreign body. Neointima formation (restenosis) and endoluminal thrombosis are associated with coronary stenting, while unstable sac occlusion and progressive dilation/fissuring of the aneurysmal wall are associated with flow diverters, but all are caused by the lack of a complete endothelial coverage of the metallic device/arterial wall which allows further activation and accumulation of blood leukocytes and platelets in the stented arterial segment. The use of “Active” stents has partially solved these issues because the associated anti-inflammatory/cytostatic drugs limit the processes leading to the proliferation of vascular stromal cells and neointima formation (restenosis). Unfortunately, their use requires a strengthening and an indefinite continuation of the antithrombotic treatment because these drugs also prevent the growth of endothelial cells and thus prevent the homeostatic endothelial covering of the stent meshes which can therefore persist in activating the platelets of the blood, even long-time (>1 year) after their implantation. Thus, the scientific and industrial community of arterial stents extensively pursues the research to identify innovative biomimetic coverings, which can prevent the local development of inflammation and thrombosis and promote endothelial cells covering allowing the integration of the medical device at the blood/vessel interface. In this context, CD31 is a very interesting biological target, because this homophilic cell-cell regulatory receptor, highly expressed by resting endothelial cells, is necessary in order to prevent the activation of circulating leukocytes and platelets. The aim of our project is to evaluate the improvement carried by directly coating stent surfaces with a CD31 biomimetic peptide, named P8RI. Preliminary data show that the covalent grafting of P8RI on superalloy sample surfaces is capable of conferring them the regulatory functions of CD31 such as survival and growth of endothelial cells and inhibition of leukocytes and platelet activation. The specific objectives of this project are: 1) to optimize the procedure of modification of the metallic surfaces and grafting of P8RI in order to be compatible with the development of an arterial stent (preservation of the physico-chemical and biological properties of the covering upon different storage and manipulation conditions); 2) to analyze the biological properties of superalloys samples (flat CoCr and 316L discs and NiTi filaments) covered with the P8RI, as compared to control samples made of the same material, in terms of growth and continuity of endothelial cells, anti-inflammatory properties and anti-thrombotic properties, in vitro; 3) to transfer the method of the modification of surface and grafting of the peptide to commercially available stents, made by the same material, and compare their biocompatibility in vivo (implantation in farm sheep coronary and rabbit carotid arteries), as assessed by histological analysis of the stented arteries, in terms of endothelial cell covering of the stent struts, degree of vascular stromal cells’ hyperplasia, signs of inflammation (leukocyte infiltration / expression of pro-inflammatory molecules) and signs of thrombosis (presence of platelets / fibrin/ plasmin).