Duchenne muscular dystrophy (DMD) is one of the most common and severe X-linked recessive disorders caused by mutations in the dystrophin gene and characterized by progressive skeletal and cardiac muscle weakness and degeneration. Although respiratory failure was previously the major cause of death, currently premature death by the second or third decade of life is mostly due to DMD-associated cardiomyopathy. To date not all the pathological mechanisms underlying this cardiomyopathy are fully elucidated and some targets, possibly exploitable for therapy, are poorly investigated. Endothelial dysfunction has been proposed to contribute to DMD pathogenesis through studies on skeletal muscle biopsies of affected subjects and on animal models; however, whether endothelial dysfunction also occurs in the heart of DMD patients and to what extent it contributes to the overall pathology remains elusive. In this Thesis project we thus take advantage of the induced Pluripotent Stem Cell (iPSC) technology, especially for the possibility to differentiate iPSCs into virtually all cell types, to generate and validate an in vitro patient-specific endothelial model of DMD-associated cardiomyopathy. Furthermore, we also studied some key features of endothelial dysfunction to verify their possible involvement in DMD-cardiomyopathy. Specifically, morphology and proliferation, endothelial-specific markers expression, mechanical stress, inflammation, oxidative stress and nitric oxide (NO) production were investigated. Our studies demonstrated that endothelial dysfunction occurred in iPSC-ECs of a 34-year-old male patient with confirmed clinical diagnosis of DMD and associated cardiomyopathy, and that the main manifestations were i) impaired expression of endothelial-specific genes, ii) pro-inflammatory phenotype, iii) oxidative stress, and iv) reduced NO production. In addition, we also differentiated patient-specific iPSCs into functional beating cardiomyocytes and cardiac fibroblasts, thus generating and validating the tools necessary to create a human multicellular cardiac model comprehensive of the main heart resident cells, that will be exploited to dissect the specific contribution of the described dysfunctional iPSC-ECs to DMD-cardiomyopathy.
La distrofia muscolare di Duchenne (DMD) è una delle più comuni e gravi malattie recessive legate all'X causata da mutazioni nel gene della distrofina e caratterizzata da progressiva debolezza e degenerazione del muscolo scheletrico e cardiaco. Sebbene in precedenza la principale causa di morte fosse l'insufficienza respiratoria, attualmente la morte prematura entro la seconda o terza decade di vita è per lo più dovuta a cardiomiopatia associata a DMD. Ad oggi non tutti i meccanismi patologici alla base di questa cardiomiopatia sono completamente chiariti ed alcuni bersagli, eventualmente sfruttabili per la terapia, sono scarsamente studiati. Attraverso studi su biopsie di muscolo scheletrico di soggetti affetti e su modelli animali, è stato proposto che la disfunzione endoteliale contribuisca alla patogenesi della DMD; tuttavia, rimane da chiarire se la disfunzione endoteliale si verifichi anche nel cuore dei pazienti con DMD e fino a che punto contribuisca alla patologia complessiva. In questo progetto di Tesi sfruttiamo quindi la tecnologia delle cellule staminali pluripotenti indotte (iPSCs), e in particolare la possibilità di differenziare le iPSCs in praticamente tutti i tipi di cellule, per generare e validare un modello endoteliale in vitro paziente-specifico di cardiomiopatia associata a DMD. Inoltre, abbiamo anche studiato alcune caratteristiche chiave della disfunzione endoteliale per verificare il loro possibile coinvolgimento nella cardiomiopatia associata a DMD. In particolare, sono stati studiati la morfologia e la proliferazione, l'espressione di marcatori endoteliali specifici, lo stress meccanico, l'infiammazione, lo stress ossidativo e la produzione di ossido nitrico (NO). I nostri studi hanno dimostrato che la disfunzione endoteliale è presente nelle cellule endoteliali da iPSCs (iPSC-ECs) di un paziente maschio di 34 anni con diagnosi clinica confermata di DMD e cardiomiopatia associata, e che le principali manifestazioni sono i) disregolata espressione di geni endoteliali specifici, ii) fenotipo pro-infiammatorio, iii) stress ossidativo e iv) ridotta produzione di NO. Inoltre, abbiamo anche differenziato le iPSCs paziente-specifiche in cardiomiociti contrattili e fibroblasti cardiaci, generando e validando così gli strumenti necessari per creare un modello cardiaco multicellulare umano comprensivo delle principali cellule residenti del cuore, che sarà sfruttato per analizzare il contributo specifico delle iPSC-ECs disfunzionali alla cardiomiopatia associata a DMD.
GENERAZIONE E CARATTERIZZAZIONE DI UN MODELLO IN VITRO DI DISFUNZIONE ENDOTELIALE IN UN PAZIENTE CON CARDIOMIOPATIA ASSOCIATA A DISTROFIA MUSCOLARE DI DUCHENNE
ZAMBONI, CHIARA
2021/2022
Abstract
Duchenne muscular dystrophy (DMD) is one of the most common and severe X-linked recessive disorders caused by mutations in the dystrophin gene and characterized by progressive skeletal and cardiac muscle weakness and degeneration. Although respiratory failure was previously the major cause of death, currently premature death by the second or third decade of life is mostly due to DMD-associated cardiomyopathy. To date not all the pathological mechanisms underlying this cardiomyopathy are fully elucidated and some targets, possibly exploitable for therapy, are poorly investigated. Endothelial dysfunction has been proposed to contribute to DMD pathogenesis through studies on skeletal muscle biopsies of affected subjects and on animal models; however, whether endothelial dysfunction also occurs in the heart of DMD patients and to what extent it contributes to the overall pathology remains elusive. In this Thesis project we thus take advantage of the induced Pluripotent Stem Cell (iPSC) technology, especially for the possibility to differentiate iPSCs into virtually all cell types, to generate and validate an in vitro patient-specific endothelial model of DMD-associated cardiomyopathy. Furthermore, we also studied some key features of endothelial dysfunction to verify their possible involvement in DMD-cardiomyopathy. Specifically, morphology and proliferation, endothelial-specific markers expression, mechanical stress, inflammation, oxidative stress and nitric oxide (NO) production were investigated. Our studies demonstrated that endothelial dysfunction occurred in iPSC-ECs of a 34-year-old male patient with confirmed clinical diagnosis of DMD and associated cardiomyopathy, and that the main manifestations were i) impaired expression of endothelial-specific genes, ii) pro-inflammatory phenotype, iii) oxidative stress, and iv) reduced NO production. In addition, we also differentiated patient-specific iPSCs into functional beating cardiomyocytes and cardiac fibroblasts, thus generating and validating the tools necessary to create a human multicellular cardiac model comprehensive of the main heart resident cells, that will be exploited to dissect the specific contribution of the described dysfunctional iPSC-ECs to DMD-cardiomyopathy.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/15685