Life expectancy has greatly increased in recent decades, and this is associated by increased incidence of age-related chronic diseases. Among several factors, oxidative stress plays a central role in the onset and progression of these pathologies. During aging, loss in antioxidant defenses can cause a pathological imbalance between scavenger systems and reactive oxygen species (ROS), which finally leads to cell damage. Indeed, among the age-related conditions, oxidative stress results to be increased in neurodegenerative disease, immune system disease, musculoskeletal disorders, frailty, and sarcopenia. In recent years, mesenchymal stem cells (MSCs), such as amniotic fluid stem cells (AFSCs), have been proposed as a potential therapy for different pathologies, thanks to their regenerative and tissue repairing capabilities. To explain their mechanisms of action different hypotheses have been proposed, including the paracrine implication of bioactive molecules carried by extracellular vesicles (EVs), such as exosomes. This thesis work demonstrated the positive effects of EVs secreted by AFSCs (AFSC-EVs) on in vitro models of different degenerative disorders which affect osteoblasts, skeletal muscles, neurons, microglia, and their functional communication. Dexamethasone (Dexa) is a synthetic glucocorticoid widely used in therapy. Despite its beneficial effects, the abuse can lead to negative consequences, including muscle loss and bone fragility. In light of this, Dexa was used to set up in vitro models of osteoporosis and muscle atrophy. First, AFSC-EVs reduced the oxidative stress increased in human osteoblast exposed to Dexa, as well as all the osteoporosis signs occurring in bone aging. Moreover, EVs ameliorated not only the differentiation capability affected in Dexa-treated bone cells, but also the pre-osteoblasts maturation potential. Despite the high plasticity of skeletal muscle tissue, during aging its regenerative potential is hampered. During muscle atrophy, neuromuscular junctions (NMJs) become vulnerable, and the impairment in this crucial region of communication between muscle cells and motor neurons (MNs) results in muscle weakness, fueling a vicious circle. In this dramatic context, how skeletal muscle sends retrograde signals to MNs through NMJs, as well as the role and source of oxidative stress, represents an intriguing field of research. To study NMJ perturbations during muscle atrophy, a MN/myotube co-culture system by microfluidic devices was set up, and the crosstalk alterations occurring during Dexa-induced muscle atrophy were investigated. AFSC-EVs presence counteracted morphological and functional defects, as well as the oxidative stress, occurring in atrophic myotubes and thus involving neurites through NMJs. Neurodegeneration characterizes several age-related disorders such as Alzheimer’s disease (AD) which presents abnormal protein aggregation, β-amyloid (Aβ) deposition, besides an increase in oxidative stress. In an animal model of AD, we observed a positive effect of AFSC-EV treatment on AD primary neuron morphology, viability, and levels of AD markers (i.e., Aβ and phospho-Tau). These effects could be due, at least in part, to the apoptotic and autophagic pathway modulation derived from the reduction in redox imbalance exerted by EVs. Since in AD, microglia plays a pivotal role in regulating the brain Aβ levels, AFSC-EV immunomodulatory properties were explored in co-cultures of AD neurons and microglia. Interestingly, EV administration mitigated the inflammatory injury caused by microglia in neurons, and significantly recovered the neurotoxicity. In conclusion, this work demonstrated that AFSC-EVs may be a potential therapeutic cell-free agent for the treatment of age-related degenerative pathologies.

L’aspettativa di vita è significativamente aumentata negli ultimi decenni e questo è stato accompagnato da un aumento dell’incidenza di patologie croniche correlate con l’età. Tra i diversi fattori, lo stress ossidativo gioca un ruolo chiave. Durante l’invecchiamento, la riduzione di difese antiossidanti porta allo squilibrio tra sistemi neutralizzanti e specie reattive dell’ossigeno (ROS), il quale conduce al danno cellulare. Infatti, è stato visto un aumento dello stress ossidativo nelle patologie neurodegenerative, immunitarie e muscoloscheletriche. Recentemente, le cellule staminali mesenchimali (MSCs), tra cui anche le cellule staminali del fluido amniotico (AFSCs), sono state proposte come possibile terapia per diverse patologie, grazie alle loro proprietà rigenerative. Per spiegare il loro meccanismo d’azione, sono state proposte diverse ipotesi e, tra queste, l’implicazione di molecole bioattive trasportate da vescicole extracellulari (EVs), come gli esosomi. Questo lavoro di tesi mostra il potenziale terapeutico di EVs secrete da AFSCs (AFSC-EVs) in modelli in vitro di diversi disordini degenerativi che possono colpire osteoblasti, muscoli scheletrici, neuroni, microglia e la loro comunicazione funzionale. Il Desametasone (Dexa) è un glucocorticoide sintetico molto utilizzato in terapia. Nonostante i suoi effetti benefici, l’abuso può portare a conseguenze negative come la perdita di tessuto muscolare e la fragilità ossea. Alla luce di ciò, il Dexa è stato utilizzato per creare modelli in vitro di osteoporosi e atrofia muscolare. Nel primo, le AFSC-EVs sono state in grado di ridurre lo stress ossidativo aumentato in osteoblasti umani esposti a Dexa, così come tutti i segni di osteoporosi caratteristici dell’invecchiamento osseo. In più, le EVs hanno potenziato il differenziamento ridotto da Dexa negli osteoblasti e nei pre-osteoblasti non trattati. Nonostante la grande plasticità del tessuto muscolare scheletrico, durante l’invecchiamento il suo potenziale rigenerativo viene ridotto. Durante l’atrofia muscolare, le giunzioni neuromuscolari (NMJs) diventano vulnerabili e l’alterazione di questa regione di comunicazione tra muscolo e motoneuroni (MNs) porta a sua volta a debolezza muscolare. Come il muscolo scheletrico invia segnali retrogradi ai MNs tramite le NMJs e il ruolo e la fonte dello stress ossidativo rappresentano un interessante ambito di ricerca. Per studiare le alterazioni delle NMJs durante l’atrofia muscolare, è stato settato un sistema di co-coltura tra MNs e miotubi mediante devices di microfluidica. Grazie a questi, sono state esaminate le alterazioni nel crosstalk in un modello di atrofia muscolare indotta da Dexa. La presenza di AFSC-EVs contrasta i difetti morfologici e funzionali, così come lo stress ossidativo, che insorgono nei miotubi atrofici e che coinvolgono i neuriti tramite le NMJs. La neurodegenerazione è presente in diversi disordini associati all’invecchiamento come l’Alzheimer (AD). L’AD è caratterizzato da una anomala aggregazione proteica, deposizione di β-amiloide (Aβ) e un aumento dello stress ossidativo. In un modello animale di AD, è stato osservato un effetto positivo sulla morfologia, vitalità e livelli dei marker di AD (Aβ e p-Tau) dei neuroni primari trattati con AFSC-EVs. Questo effetto può essere dovuto alla modulazione di apoptosi e autofagia derivante dalla riduzione dello squilibrio redox esercitata dalle EVs. Infine, considerato il ruolo chiave della microglia nella regolazione dei livelli di Aβ in questa patologia, le proprietà immuno-modulatorie delle EVs sono state approfondite in un modello di co-coltura tra neuroni e microglia. La somministrazione di EVs ha mitigato il danno infiammatorio causato dalla microglia sui neuroni, riducendo significativamente la neurotossicità.

Ruolo degli esosomi derivanti dalle cellule staminali del fluido amniotico nel contrastare i disordini correlati con l'invecchiamento / Martina Gatti , 2023 May 23. 35. ciclo, Anno Accademico 2021/2022.

Ruolo degli esosomi derivanti dalle cellule staminali del fluido amniotico nel contrastare i disordini correlati con l'invecchiamento

GATTI, MARTINA
2023

Abstract

Life expectancy has greatly increased in recent decades, and this is associated by increased incidence of age-related chronic diseases. Among several factors, oxidative stress plays a central role in the onset and progression of these pathologies. During aging, loss in antioxidant defenses can cause a pathological imbalance between scavenger systems and reactive oxygen species (ROS), which finally leads to cell damage. Indeed, among the age-related conditions, oxidative stress results to be increased in neurodegenerative disease, immune system disease, musculoskeletal disorders, frailty, and sarcopenia. In recent years, mesenchymal stem cells (MSCs), such as amniotic fluid stem cells (AFSCs), have been proposed as a potential therapy for different pathologies, thanks to their regenerative and tissue repairing capabilities. To explain their mechanisms of action different hypotheses have been proposed, including the paracrine implication of bioactive molecules carried by extracellular vesicles (EVs), such as exosomes. This thesis work demonstrated the positive effects of EVs secreted by AFSCs (AFSC-EVs) on in vitro models of different degenerative disorders which affect osteoblasts, skeletal muscles, neurons, microglia, and their functional communication. Dexamethasone (Dexa) is a synthetic glucocorticoid widely used in therapy. Despite its beneficial effects, the abuse can lead to negative consequences, including muscle loss and bone fragility. In light of this, Dexa was used to set up in vitro models of osteoporosis and muscle atrophy. First, AFSC-EVs reduced the oxidative stress increased in human osteoblast exposed to Dexa, as well as all the osteoporosis signs occurring in bone aging. Moreover, EVs ameliorated not only the differentiation capability affected in Dexa-treated bone cells, but also the pre-osteoblasts maturation potential. Despite the high plasticity of skeletal muscle tissue, during aging its regenerative potential is hampered. During muscle atrophy, neuromuscular junctions (NMJs) become vulnerable, and the impairment in this crucial region of communication between muscle cells and motor neurons (MNs) results in muscle weakness, fueling a vicious circle. In this dramatic context, how skeletal muscle sends retrograde signals to MNs through NMJs, as well as the role and source of oxidative stress, represents an intriguing field of research. To study NMJ perturbations during muscle atrophy, a MN/myotube co-culture system by microfluidic devices was set up, and the crosstalk alterations occurring during Dexa-induced muscle atrophy were investigated. AFSC-EVs presence counteracted morphological and functional defects, as well as the oxidative stress, occurring in atrophic myotubes and thus involving neurites through NMJs. Neurodegeneration characterizes several age-related disorders such as Alzheimer’s disease (AD) which presents abnormal protein aggregation, β-amyloid (Aβ) deposition, besides an increase in oxidative stress. In an animal model of AD, we observed a positive effect of AFSC-EV treatment on AD primary neuron morphology, viability, and levels of AD markers (i.e., Aβ and phospho-Tau). These effects could be due, at least in part, to the apoptotic and autophagic pathway modulation derived from the reduction in redox imbalance exerted by EVs. Since in AD, microglia plays a pivotal role in regulating the brain Aβ levels, AFSC-EV immunomodulatory properties were explored in co-cultures of AD neurons and microglia. Interestingly, EV administration mitigated the inflammatory injury caused by microglia in neurons, and significantly recovered the neurotoxicity. In conclusion, this work demonstrated that AFSC-EVs may be a potential therapeutic cell-free agent for the treatment of age-related degenerative pathologies.
Role of exosomes derived from amniotic fluid stem cells in counteracting age-related disorders
23-mag-2023
PALUMBO, Carla
MARALDI, Tullia
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/1305468
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