Évaluation de l'efficacité neuroprotectrice et neuro-restauratrice du lysat de plaquettes humaines chauffe dans des modèles de lésions traumatiques cérébrales

Traumatic brain injury (TBI) remains a global health challenge nowadays, impacting over 50 million people per year globally. This situation is partly linked to the fact that TBI is among the central nervous system disorders whose management mostly requires long-term care. It incurs a substantial economic burden to health systems and costing the global economy more than 400 million dollars. In either high, middle, or low-income countries, TBI is associated with significant economic and societal changes that deserve attention. The disease is described as one of the most complexes, inducing some disproportionate effects between the countries. Unfortunately, the intervention strategies are still facing several limitations at the global level despite all the health sciences’ progress. These obstacles are the surge of neuroinflammation, leading to progressive neuronal degeneration and cognitive deficit. Efforts are made to stop this “silent killer”, but there is a failure to manage the long-term burden of TBI efficiently until now.Nowadays, there is growing evidence that platelet lysates are full of bioactive compounds, and they could constitute a powerful natural neuroprotective agent. Few studies have already shown their therapeutic potential in stroke, amyotrophic lateral sclerosis, and Parkinson's disease. Thus, we hypothesized that the delivery of human platelet lysate at an injured area in the brain could provide a suitable environment for recovery.The current project is intending to develop an innovative approach for the treatment of TBI. We aim to give the proof-of-concept of the interest of using heat-treated human platelet pellet lysate (HPPL) as a neuroprotective agent in TBI using experimental models.We used cells and animal models of TBI to achieve our goal. We first prepared HPPL from non-pathogen-reduced platelet concentrates (PCs) and pathogen-inactivated PCs (I-HPPL) according to a previously established procedure. We evaluated their safety and functionality using cell models relevant to TBI, including viability assays, wound healing, anti-inflammatory activity, protein expressions, and anti-ferroptosis effect. The safety assessment of the platelet biomaterial was done using neuronal and endothelial cells and its neuroprotective potential with primary neurons, dopaminergic cells line and, a ferroptosis inducer.Mouse TBI models were used to assess the therapeutic potential of HPPL. We targeted it impact on motor function, neuroinflammation, oxidative stress, and synaptic loss. Behavior tests, gene expression, fluorescent staining, ELISA, Western blot, and proteomics have been used during the investigation. The in vitro experiment performed to investigate the platelet lysate’s safety demonstrated clearly that HPPL/I-HPPL contain bioactive molecules and did not affect cell’s viability or induced stress. Moreover, HPPL and I-HPPL did not affect synaptic and neuronal protein expression and revealed anti-ferroptosis potential. This finding leads to further investigation of HPPL's beneficial effect in vivo. HPPL administration to TBI mice improved their motor function, mitigated the inflammation and oxidative stress. HPPL also decreased the synaptic proteins lost.HPPL is safe and exerted neuroprotective activity in vitro. It successfully reversed the motor deficit, inflammation, and stress triggered by brain injury in mice.Les lésions cérébrales traumatiques (LCT) restent un défi de santé publique de nos jours car celles-ci touchent plus de cinquante millions de personnes dans le monde par an. Leur prise en charge nécessite généralement des soins de longue durée et ceci engendre malheureusement des dépenses financières importantes estimées à plus de 400 millions de dollars US par an. Les LCT font partie des plus complexes traumatismes affectant le cerveau humain. Dans les cas chroniques par exemple, l’une des complications des LCT est la persistance de la neuroinflammation qui conduit inévitablement à une dégénérescence neuronale et à un déficit cognitif. Malheureusement, les stratégies d'intervention se heurtent encore à plusieurs obstacles malgré les progrès technologiques actuels. Des efforts sont faits pour arrêter ce « tueur silencieux », mais jusqu'à nos jours, aucune stratégie ne s’est avérée efficace contre les perturbations physiologiques à long terme des LCT.De nos jours, il apparaît de plus en plus clairement que le lysat plaquettaire contient une pléthore de molécules actives pouvant exercer des activités neuroprotectrices. Ce produit neuroprotecteur naturel pourrait donc être bénéfique dans le traitement des maladies neurodégénératives comme l’ont montré des études antérieures avec des modèles expérimentaux d’accidents vasculaires cérébraux, de sclérose latérale amyotrophique et de la maladie de Parkinson.Nous avons émis l'hypothèse que l’injection de ce lysat plaquettaire dans une zone lésée du cerveau favoriserait la cicatrisation et réduirait la mort neuronale.Notre projet avait donc pour but de développer une approche innovante pour le traitement de la lésion traumatique cérébrale. Nous visions à donner la preuve du concept de l'intérêt d’utiliser le lysat chauffé de plaquettes humaines (HPPL) comme agent neuroprotecteur.Du HPPL a été préparé à partir de concentrés plaquettaires non viro-inactivés et viro-inactivés selon une procédure préalablement établie. L’impact sur la viabilité des cellules, l’absence d’activité pro-inflammatoire, le potentiel de stimuler la cicatrisation du HPPL ont été évalué à l'aide de modèles cellulaires pertinents. L’activité d’inhibition de la ferroptose par le HPPL a été aussi investiguée en utilisant des cellules de LUHMES et des cultures primaires de neurones. En fin, des modèles de LCT de souris ont été utilisés pour évaluer le potentiel « neuro-restaurateur » du HPPL. Nous avons ciblé ici son impact sur la fonction motrice, la neuroinflammation, le stress oxydatif et la perte synaptique. Des tests de comportement, d'expression de gènes, d’histologie, d'ELISA, de Western blot et d’analyse protéomique ont été utilisés au cours de cette étude.L'étude in vitro a révélé que les HPPL contiennent une pléthore de molécules bioactives qui sont non-toxiques pour les cellules, ni n’induisent de stress aux cellules traitées. Bien plus, les HPPL n'ont pas exercé d’impact négatif sur l'expression des protéines synaptiques et neuronales, et ont conservé leur potentiel « anti-ferroptose ». L'administration du HPPL à des souris ayant des LCT, a amélioré leur fonction motrice, atténué l'inflammation et le stress oxydatif et réduit la perte synaptique.En résume, le HPPL/I-HPPL possède une activité neuroprotectrice révélée à travers les essais in vitro. In vivo, il a réussi à inverser le déficit moteur, moduler l’activation des cellules gliales et le stress oxydatif provoqués par la LCT induite aux souris

Can the administration of platelet lysates to the brain help treat neurological disorders?

International audienceNeurodegenerative disorders of the central nervous system (CNS) and brain traumatic insults are characterized by complex overlapping pathophysiological alterations encompassing neuroinflammation, alterations of synaptic functions, oxidative stress, and progressive neurodegeneration that eventually lead to irreversible motor and cognitive dysfunctions. A single pharmacological approach is unlikely to provide a complementary set of molecular therapeutic actions suitable to resolve these complex pathologies. Recent preclinical data are providing evidence-based scientific rationales to support biotherapies based on administering neurotrophic factors and extracellular vesicles present in the lysates of human platelets collected from healthy donors to the brain. Here, we present the most recent findings on the composition of the platelet proteome that can activate complementary signaling pathways in vivo to trigger neuroprotection, synapse protection, anti-inflammation, antioxidation, and neurorestoration. We also report experimental data where the administration of human platelet lysates (HPL) was safe and resulted in beneficial neuroprotective effects in established rodent models of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, traumatic brain injury, and stroke. Platelet-based biotherapies, prepared from collected platelet concentrates (PC), are emerging as a novel pragmatic and accessible translational therapeutic strategy for treating neurological diseases. Based on this assumption, we further elaborated on various clinical, manufacturing, and regulatory issues that need to be addressed to ensure the ethical supply, quality, and safety of HPL preparations for treating neurodegenerative and traumatic pathologies of the CNS. HPL made from PC may become a unique approach for scientifically based treatments of neurological disorders readily accessible in low-, middle-, and high-income countries

The role of adenosine A2A receptors in Alzheimer's disease and tauopathies

International audienceAdenosine signals through four distinct G protein-coupled receptors that are located at various synapses, cell types and brain areas. Through them, adenosine regulates neuromodulation, neuronal signaling, learning and cognition as well as the sleep-wake cycle, all strongly impacted in neurogenerative disorders, among which Alzheimer's Disease (AD). AD is a complex form of cognitive deficits characterized by two pathological hallmarks: extracellular deposits of aggregated β-amyloid peptides and intraneuronal fibrillar aggregates of hyper- and abnormally phosphorylated Tau proteins. Both lesions contribute to the early dysfunction and loss of synapses which are strongly associated to the development of cognitive decline in AD patients. The present review focuses on the pathophysiological impact of the A2ARs dysregulation observed in cognitive area from AD patients. We are reviewing not only evidence of the cellular changes in A2AR levels in pathological conditions but also describe what is currently known about their consequences in term of synaptic plasticity, neuro-glial miscommunication and memory abilities. We finally summarize the proof-of-concept studies that support A2AR as credible targets and the clinical interest to repurpose adenosine drugs for the treatment of AD and related disorders. This article is part of the Special Issue on "Purinergic Signaling: 50 years"

Characterization and Chromatographic Isolation of Platelet Extracellular Vesicles from Human Platelet Lysates for Applications in Neuroregenerative Medicine

International audienceHuman platelet lysates (HPLs) made from clinical-grade platelet concentrates are currently evaluated in the preclinical models of Parkinson's disease, Alzheimer's disease, traumatic brain injury, and others, as a new polyvalent neuroprotective biotherapy of the central nervous system. However, the presence and content of extracellular vesicles (EVs) in HPLs and their potential contribution to the neuroprotective and neurorestorative activities of HPLs are still unknown. We, therefore, characterized the EVs present in four different HPL preparations and after purification by size-exclusion chromatography. We then tested the effect of the isolated EVs on neuronal cell repair. We identified that all four HPLs contained a high and similar amount of EVs (1011 to 1012/mL) with a mean size ranging from ca. 50 to 300 nm and a negative zeta potential as determined by nanoparticle tracking analysis and dynamic light scattering. Western blot analysis revealed that the EVs present in HPLs expressed the clusters of differentiation 41 (CD41) and 61 (CD61) characteristic of platelets. These EVs were efficiently isolated from HPL proteins by Sepharose CL-2B size-exclusion column chromatography as confirmed by total protein determination and protein profile by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with 73-85% recovery and maintenance of their size, negative zeta potential, and CD41 and CD61 expression. Interestingly, the EVs purified from the four HPLs exhibited a differential capacity to promote cell growth and migration in a wound-healing assay using SH-SY5Y neuronal cells, and one EV preparation stimulated network formation in primary neuronal cultures. These data indicated that the EVs present in HPLs have different neuroregenerative capacities and that some EV preparations may have interesting applications as a stand-alone therapy for usage in neuroregenerative medicine

Heat-treated human platelet pellet lysate modulates microglia activation, favors wound healing and promotes neuronal differentiation in vitro

International audienceThe neuro-restorative efficacy of human platelet lysates in neurodegenerative disorders is still under investigation. Platelets prepared from standard and pathogen reduced platelet concentrates were pelletized, washed, concentrated, and subjected to freeze-thawing. The lysate was heated to 56°C for 30 min and characterized. Toxicity was evaluated using SH- SY5Y neuroblastoma, BV-2 microglial, and EA-hy926 endothelial cells. Inflammatory activity was tested by examining tumor necrosis factor (TNF) and cyclooxygenase (COX)-2 expres- sions by BV2 microglia with or without stimulation by lipopolysaccharides (LPS). The capacity to stimulate wound healing was evaluated by a scratch assay, and the capacity to differentiate SH-SY5Y into neurons was also examined. Platelet lysates contained a range of neurotrophins. They were not toxic to SH-SY5Y, EA-hy926, or BV-2 cells, did not induce the expression of TNF or COX-2 inflammatory markers by BV2 microglia, and decreased inflammation after LPS stimulation. They stimulated the wound closure in the scratch assay and induced SH-SY5Y differentiation as revealed by the increased length of neurites as well as β3-tubulin and neurofilament staining. These data confirm the ther- apeutic potential of platelet lysates in the treatment of disorders of the central nervous system and support further evaluation as novel neurorestorative biotherapy in preclinical models

Human platelet lysates for human cell propagation

A pathogen-free and standardized xeno-free supplement of growth media is required for the ex vivo propagation of human cells used as advanced therapeutic medicinal products and for clinical translation in regenerative medicine and cell therapies. Human platelet lysate (HPL) made from therapeutic-grade platelet concentrate (PC) is increasingly regarded as being an efficient xeno-free alternative growth medium supplement to fetal bovine serum (FBS) for clinical-grade isolation and/or propagation of human cells. Most experimental studies establishing the superiority of HPL over FBS were conducted using mesenchymal stromal cells (MSCs) from bone marrow or adipose tissues. Data almost unanimously concur that MSCs expanded in a media supplemented with HPL have improved proliferation, shorter doubling times, and preserved clonogenicity, immunophenotype, in vitro trilineage differentiation capacity, and T-cell immunosuppressive activity. HPL can also be substituted for FBS when propagating MSCs from various other tissue sources, including Wharton jelly, the umbilical cord, amniotic fluid, dental pulp, periodontal ligaments, and apical papillae. Interestingly, HPL xeno-free supplementation is also proving successful for expanding human-differentiated cells, including chondrocytes, corneal endothelium and corneal epithelium cells, and tenocytes, for transplantation and tissue-engineering applications. In addition, the most recent developments suggest the possibility of successfully expanding immune cells such as macrophages, dendritic cells, and chimeric antigen receptor-T cells in HPL, further broadening its use as a growth medium supplement. Therefore, strong scientific rationale supports the use of HPL as a universal growth medium supplement for isolating and propagating therapeutic human cells for transplantation and tissue engineering. Efforts are underway to ensure optimal standardization and pathogen safety of HPL to secure its reliability for clinical-grade cell-therapy and regenerative medicine products and tissue engineering

Platelet extracellular vesicles are efficient delivery vehicles of doxorubicin, an anti-cancer drug: preparation and in vitro characterization

Platelet extracellular vesicles (PEVs) are an emerging delivery vehi for anticancer drugs due to their ability to target and remain in the tumor microenvironment. However, there is still a lack of understanding regarding yields, safety, drug loading efficiencies, and efficacy of PEVs. In this study, various methods were compared to generate PEVs from clinical-grade platelets, and their properties were examined as vehicles for doxorubicin (DOX). Sonication and extrusion produced the most PEVs, with means of 496 and 493 PEVs per platelet (PLT), respectively, compared to 145 and 33 by freeze/thaw and incubation, respectively. The PEVs were loaded with DOX through incubation and purified by chromatography. The size and concentration of the PEVs and PEV-DOX were analyzed using dynamic light scattering and nanoparticle tracking analysis. The results showed that the population sizes and concentrations of PEVs and PEV-DOX were in the ranges of 120–150 nm and 1.2–6.2 × 1011 particles/mL for all preparations. The loading of DOX determined using fluorospectrometry was found to be 2.1 × 106, 1.7 × 106, and 0.9 × 106 molecules/EV using freeze/thaw, extrusion, and sonication, respectively. The internalization of PEVs was determined to occur through clathrin-mediated endocytosis. PEV-DOX were more efficiently taken up by MDA-MB-231 breast cancer cells compared to MCF7/ADR breast cancer cells and NIH/3T3 cells. DOX-PEVs showed higher anticancer activity against MDA-MB-231 cells than against MCF7/ADR or NIH/3T3 cells and better than acommercial liposomal DOX formulation. In conclusion, this study demonstrates that PEVs generated by PLTs using extrusion, freeze/thaw, or sonication can efficiently load DOX and kill breast cancer cells, providing a promising strategy for further evaluation in preclinical animal models. The study findings suggest that sonication and extrusion are the most efficient methods to generate PEVs and that PEVs loaded with DOX exhibit significant anticancer activity against MDA-MB-231 breast cancer cells

The neuroprotective activity of heat-treated human platelet lysate biomaterials manufactured from outdated pathogen-reduced (amotosalen/UVA) platelet concentrates

Background: Effective neurorestorative therapies of neurodegenerative diseases must be developed. There is increasing interest in using human platelet lysates, rich in neurotrophic factors, as novel disease-modifying strategy of neurodegeneration. To ensure virus safety, pathogen reduction treatments should be incorporated in the preparation process of the platelet concentrates used as source material. We therefore investigated whether platelet concentrates (PC) pathogen-inactivated using a licensed photo-inactivation treatment combining photosensitive psoralen (amotosalen) and UVA irradiation (Intercept) can serve as source material to prepare platelet lysates with preserved neuroprotective activity in Parkinson's disease models. Methods: Intercept treated-PCs were centrifuged, when reaching expiry day (7 days after collection), to remove plasma and platelet additive solution. The platelet pellet was re-suspended and concentrated in phosphate buffer saline, subjected to 3 freeze-thaw cycles (-80 degrees C/37 degrees C) then centrifuged to remove cell debris. The supernatant was recovered and further purified, or not, by heat-treatment as in our previous investigations. The content in proteins and neurotrophic factors was determined and the toxicity and neuroprotective activity of the platelet lysates towards LUHMES cells or primary cortical/hippocampal neurons were assessed using ELISA, flow cytometry, cell viability and cytotoxicity assays and proteins analysis by Western blot. Results: Platelet lysates contained the expected level of total proteins (ca. 7-14 mg/mL) and neurotrophic factors. Virally inactivated and heat-treated platelet lysates did not exert detectable toxic effects on neither Lund human mesencephalic dopaminergic LUHMES cell line nor primary neurons. When used at doses of 5 and 0.5%, they enhanced the expression of tyrosine hydroxylase and neuron-specific enolase in LUHMES cells and did not significantly impact synaptic protein expression in primary neurons, respectively. Furthermore, virally-inactivated platelet lysates tested were found to exert very strong neuroprotection effects on both LUHMES and primary neurons exposed to erastin, an inducer of ferroptosis cell death. Conclusion: Outdated Intercept pathogen-reduced platelet concentrates can be used to prepare safe and highly neuroprotective human heat-treated platelet pellet lysates. These data open reassuring perspectives in the possibility to develop an effective biotherapy using virally-inactivated platelet lysates rich in functional neurotrophins for neuroregenerative medicine, and for further bio-industrial development. However, the data should be confirmed in animal models

Neuroprotective activity of a virus‐safe nanofiltered human platelet lysate depleted of extracellular vesicles in Parkinson's disease and traumatic brain injury models

International audienceAbstract Brain administration of human platelet lysates (HPL) is an emerging biotherapy of neurodegenerative and traumatic diseases of the central nervous system (CNS). HPLs being prepared from pooled platelet concentrates (PCs) increasing viral risks, manufacturing processes should incorporate robust virus-reduction treatments. We evaluated a 19±2-nm virus removal nanofiltration process using hydrophilic regenerated cellulose hollow fibers on the properties of a neuroprotective heat-treated HPL (HPPL). Spiking experiments demonstrated >5.30 log removal of 20~22-nm non-enveloped minute virus of mice-mock particles using an immuno-quantitative polymerase chain reaction assay. The nanofiltered HPPL (NHPPL) contained a range of neurotrophic factors like HPPL. There was >2 log removal of extracellular vesicles (EVs), associated with decreased expression of pro-thrombogenic phosphatidylserine and procoagulant activity. LC-MS/MS proteomics showed that ca. 80% of HPPL proteins, including neurotrophins, cytokines, and antioxidants, were still found in NHPPL, whereas proteins associated with some infections and cancer-associated pathways, pro-coagulation and EVs, were removed. NHPPL maintained intact neuroprotective activity in LUHMES dopaminergic neuron model of Parkinson’s disease (PD), stimulated the differentiation of SH-SY5Y neuronal cells and showed preserved anti-inflammatory function upon intranasal administration in a mouse model of traumatic brain injury (TBI). Therefore, nanofiltration of HPL is feasible, lowers the viral, prothrombotic and procoagulant risks, and preserves the neuroprotective and anti-inflammatory properties in neuronal pre-clinical of PD and TBI

Human platelet lysate biotherapy for traumatic brain injury: preclinical assessment

International audienceTraumatic brain injury leads to major brain anatomopathological damages underlined by neuroinflammation, oxidative stress and progressive neurodegeneration, ultimately leading to motor and cognitive deterioration. The multiple pathological events resulting from traumatic brain injury can be addressed not by a single therapeutic approach, but rather by a synergistic biotherapy capable of activating a complementary set of signaling pathways and providing synergistic neuroprotective, anti-inflammatory, antioxidative, and neurorestorative activities. Human platelet lysate might fulfill these requirements as it is comprised of a plethora of biomolecules readily accessible as a traumatic brain injury biotherapy. In the present study, we have tested the therapeutic potential of human platelet lysate using in vitro and in vivo models of traumatic brain injury. We first prepared and characterized platelet lysate from clinical-grade human platelet concentrates. Platelets were pelletized, lysed by three freeze-thaw cycles, and centrifuged. The supernatant was purified by 56 °C-30 minutes heat-treatment and spun to obtain the heat-treated platelet pellet lysate that was characterized by ELISA and proteomic analyses. Two mouse models were used to investigate platelet lysate neuroprotective potential. The injury was induced by an in-house manual controlled scratching of the animals' cortex or by controlled cortical impact injury. The platelet lysate treatment was performed by topical application of 60 µL in the lesioned area, followed by daily 60 µL intranasal administration from day 1 to 6 post-injury. Platelet lysate proteomics identified over 1000 proteins including growth factors, neurotrophins, and antioxidants. ELISA detected several neurotrophic and angiogenic factors at ca. 1-50 ng/mL levels. We demonstrate, using the two mouse models of traumatic brain injury that topical application and intranasal platelet lysate consistently improved mice motor function in the beam and rotarod tests, mitigated cortical neuroinflammation, and oxidative stress in the injury area, as revealed by downregulation of pro-inflammatory genes and the reduction in reactive oxygen species levels. Moreover, platelet lysate treatment reduced the loss of cortical synaptic proteins. Unbiased proteomic analyses revealed that HPPL reversed several pathways promoted by both CCI and CBS and related to transport, post-synaptic density, mitochondria or lipid metabolism. The present data strongly support for the first time that human platelet lysate is a reliable and effective therapeutic source of neurorestorative factors. Therefore, brain administration of platelet lysate is a therapeutical strategy that deserves serious and urgent consideration for universal brain trauma treatment