RESUMEN
Intracerebral haemorrhage is an unmet medical need affecting more than 3 million people worldwide every year and leading to the formation of an intracerebral haematoma. Updated guidelines (2022) for the management of intracerebral haemorrhage patients recognize that minimally invasive approaches for the evacuation of supratentorial intracerebral haemorrhage have demonstrated reductions in mortality compared with medical management alone. However, improvement of functional outcome with a procedure involving thrombolytic therapy was neutral in the last large phase 3 clinical trial and requires a more effective and safer thrombolytic agent than those currently available. Here, we demonstrate that O2L-001 allows for the extended release of W253R/R275S recombinant tissue-type plasminogen activator (rtPA). A new rtPA variant, called optimized tPA (OptPA), offers improved efficacy for haematoma evacuation as well as improved safety. OptPA was produced in a Chinese hamster ovary cell line before purification, nanoprecipitation using the NANOp2Lysis® technological platform followed by suspension in a solution of 17% poloxamer 407 to obtain O2L-001. Plasmin generation assays were performed to demonstrate O2L-001 safety. Ex vivo haematoma models using human blood were used to demonstrate O2L-001 thrombolysis properties and efficacy. For the best translational significance, a clinical sized haematoma was used to ensure catheter placement and to allow administration of the thrombolytic agent into the core of the haematoma via a minimally invasive procedure. The capacity of OptPA to convert plasminogen into plasmin is strongly decreased compared to rtPA, thereby reducing potential bleeding events. However, a clot lysis assay showed that OptPA had the same fibrinolytic activity as rtPA. We demonstrated that long-term exposure to a thrombolytic agent was essential to achieve high thrombolysis efficacy. Indeed, 24 h continuous exposure to 0.1 µg/ml rtPA had similar efficacy than repeated short exposure to 30 µg/ml rtPA. This finding led to the development of O2L-001, allowing the extended release of OptPA in the first 6 h following injection. An ex vivo model using human blood was used to demonstrate O2L-001 efficacy. Interestingly, unlike rtPA, O2L-001 was able to induce the complete lysis of the 5 ml haematoma. In clinical sized haematomas (obtained from 30 ml of human blood), a single injection of O2L-001 at 1 mg/ml into the core of the haematoma led to a 44% increase in thrombolysis compared to rtPA. Taken together, these results demonstrate that O2L-001 provides new hope for haematoma evacuation and the treatment of patients with intracerebral haemorrhage.
Asunto(s)
Fibrinolisina , Fibrinolíticos , Animales , Cricetinae , Humanos , Fibrinolíticos/uso terapéutico , Fibrinolisina/uso terapéutico , Células CHO , Cricetulus , Activador de Tejido Plasminógeno/uso terapéutico , Hemorragia Cerebral/tratamiento farmacológico , Terapia Trombolítica , Hematoma/tratamiento farmacológicoRESUMEN
Cerebral ischemia is a pathology involving a cascade of cellular mechanisms, leading to the deregulation of proteostasis, including macroautophagy/autophagy, and finally to neuronal death. If it is now accepted that cerebral ischemia induces autophagy, the effect of thrombolysis/energy recovery on proteostasis remains unknown. Here, we investigated the effect of thrombolysis by PLAT/tPA (plasminogen activator, tissue) on autophagy and neuronal death. In two in vitro models of hypoxia reperfusion and an in vivo model of thromboembolic stroke with thrombolysis by PLAT/tPA, we found that ischemia enhances neuronal deleterious autophagy. Interestingly, PLAT/tPA decreases autophagy to mediate neuroprotection by modulating the PI3K-AKT-MTOR pathways both in vitro and in vivo. We identified IGF1R (insulin-like growth factor I receptor; a tyrosine kinase receptor) as the effective receptor and showed in vitro, in vivo and in human stroke patients and that PLAT/tPA is able to degrade IGFBP3 (insulin-like growth factor binding protein 3) to increase IGF1 (insulin-like growth factor 1) bioavailability and thus IGF1R activation.Abbreviations: AKT/protein kinase B: thymoma viral proto-oncogene 1; EGFR: epidermal growth factor receptor; Hx: hypoxia; IGF1: insulin-like growth factor 1; IGF1R: insulin-like growth factor I receptor; IGFBP3: insulin-like growth factor binding protein 3; Ka: Kainate; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK/ERK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; OGD: oxygen and glucose deprivation; OGDreox: oxygen and glucose deprivation + reoxygentation; PepA: pepstatin A1; PI3K: phosphoinositide 3-kinase; PLAT/tPA: plasminogen activator, tissue; PPP: picropodophyllin; SCH77: SCH772984; ULK1: unc-51 like kinase 1; Wort: wortmannin.
Asunto(s)
Isquemia Encefálica , Accidente Cerebrovascular , Autofagia , Isquemia Encefálica/tratamiento farmacológico , Glucosa/farmacología , Humanos , Hipoxia , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Oxígeno/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Accidente Cerebrovascular/tratamiento farmacológico , Serina-Treonina Quinasas TOR/metabolismo , Terapia Trombolítica , Activador de Tejido Plasminógeno/metabolismo , Activador de Tejido Plasminógeno/farmacologíaRESUMEN
Tissue-type plasminogen activator (tPA) plays roles in the development and the plasticity of the nervous system. Here, we demonstrate in neurons, that by opposition to the single chain form (sc-tPA), the two-chains form of tPA (tc-tPA) activates the MET receptor, leading to the recruitment of N-Methyl-d-Aspartate receptors (NMDARs) and to the endocytosis and proteasome-dependent degradation of NMDARs containing the GluN2B subunit. Accordingly, tc-tPA down-regulated GluN2B-NMDAR-driven signalling, a process prevented by blockers of HGFR/MET and mimicked by its agonists, leading to a modulation of neuronal death. Thus, our present study unmasks a new mechanism of action of tPA, with its two-chains form mediating a crosstalk between MET and the GluN2B subunit of NMDARs to control neuronal survival.
Asunto(s)
Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Feto , Ratones , Cultivo Primario de Células , Isoformas de Proteínas , Proteínas Proto-Oncogénicas c-met/fisiología , Receptor Cross-Talk/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal , Activador de Tejido Plasminógeno/fisiologíaRESUMEN
Cerebral ischemia is a complex pathology involving a cascade of cellular mechanisms, which deregulate proteostasis and lead to neuronal death. Proteostasis refers to the equilibrium between protein synthesis, folding, transport, and protein degradation. Within the brain proteostasis plays key roles in learning and memory by controlling protein synthesis and degradation. Two important pathways are implicated in the regulation of proteostasis: the unfolded protein response (UPR) and macroautophagy (called hereafter autophagy). Both are necessary for cell survival, however, their over-activation in duration or intensity can lead to cell death. Moreover, UPR and autophagy can activate and potentiate each other to worsen the issue of cerebral ischemia. A better understanding of autophagy and ER stress will allow the development of therapeutic strategies for stroke, both at the acute phase and during recovery. This review summarizes the latest therapeutic advances implicating ER stress or autophagy in cerebral ischemia. We argue that the processes governing proteostasis should be considered together in stroke, rather than focusing either on ER stress or autophagy in isolation.
RESUMEN
Although recent technical advances in thrombectomy have revolutionised acute stroke treatment, prevalence of disability and death related to stroke remain high. Therefore, plasminogen activators-eukaryotic, bacterial, or engineered forms that can promote fibrinolysis by converting plasminogen into active plasmin and facilitate clot breakdown-are still commonly used in the acute treatment of ischaemic stroke. Hence, plasminogen activators have become a crucial area for clinical investigation for their ability to recanalise occluded arteries in ischaemic stroke and to accelerate haematoma clearance in haemorrhagic stroke. However, inconsistent results, insufficient evidence of efficacy, or reports of side-effects in trial settings might reduce the use of plasminogen activators in clinical practice. Additionally, the mechanism of action for plasminogen activators could extend beyond the vessel lumen and involve plasminogen-independent processes, which would suggest that plasminogen activators have also non-fibrinolytic roles. Understanding the complex mechanisms of action of plasminogen activators can guide future directions for therapeutic interventions in patients with stroke.
Asunto(s)
Fibrinólisis/efectos de los fármacos , Accidente Cerebrovascular/terapia , Activador de Tejido Plasminógeno/farmacología , Activador de Tejido Plasminógeno/uso terapéutico , Animales , HumanosRESUMEN
The unfolded protein response (UPR) is an endoplasmic reticulum (ER) -related stress conserved pathway that aims to protect cells from being overwhelmed. However, when prolonged, UPR activation converts to a death signal, which relies on its PERK-eIF2α branch. Overactivation of the UPR has been implicated in many neurological diseases, including cerebral ischaemia. Here, by using an in vivo thromboembolic model of stroke on transgenic ER stress-reporter mice and neuronal in vitro models of ischaemia, we demonstrate that ischaemic stress leads to the deleterious activation of the PERK branch of the UPR. Moreover, we show that the serine protease tissue-type plasminogen activator (tPA) can bind to cell surface Grp78 (78 kD glucose-regulated protein), leading to a decrease of the PERK pathway activation, thus a decrease of the deleterious factor CHOP, and finally promotes neuroprotection. Altogether, this work highlights a new role and a therapeutic potential of the chaperone protein Grp78 as a membrane receptor of tPA capable to prevent from ER stress overactivation.