During my educational path as a PhD Student, I have contributed to the realization of different research projects, all related to the study of atherosclerosis and cholesterol metabolism through the use of animal models. Among these, I focused my PhD thesis mainly on two of the studies I co-authored, one as first author. Moreover, a third study, still ongoing and whose results have been presented at National and International conferences, have been discussed. Specifically, my thesis includes a study of the effect of a salmon protein hydrolysate on atherosclerosis development in ApoE-deficient mice, on the basis of previous evidences indicating that fish protein peptides and hydrolysates are able to exert beneficial effects on cardiovascular risk factors. Mice fed a high-fat diet with 5% salmon protein hydrolysate for 12 weeks showed a significantly reduced atherosclerotic plaque area in the aortic arch and aortic sinus versus controls. The effect of this dietary supplementation was shown to be mediated by the inhibition of systemic inflammation activation and it seemed to be independent of changes in plasma lipid levels. Moreover, I described and discussed the investigation of the effect of L-homoarginine, an L-arginine homologue derived from lysine, on neointimal hyperplasia, a pathological condition which can occur at sites of subclinical atherosclerosis or after angioplasty interventions. Data from clinical studies indicated L-homoarginine as a novel biomarker for cardiovascular diseases. In particular, two independent Genome-Wide Association Studies identified an association between L-homoarginine plasma levels and SNPs related to the arginine:glycine amidinotransferase gene, which plays a central role in energy metabolism, being involved in homoarginine and, indirectly, creatine synthesis. For the first time, we have demonstrated that in vivo administration of homoarginine in a rat model of carotid balloon injury was able to inhibit neointimal formation, at least in part, by inhibiting vascular cell proliferation and by increasing both arginine availability and nitric oxide production, suggesting homoarginine as a potential therapeutical option for cardiovascular protection. Finally, my thesis also includes the ongoing characterization of microRNA expression patterns in tissues involved in cholesterol metabolism and atherosclerosis, harvested from Ldlr-deficient, Pcsk9-deficient and wild-type C57BL/6 mice after chow or a high fat feeding. We have identified the microRNAs which resulted differentially expressed (DE) in our experimental conditions by high-throughput sequencing and bioinformatics analyses. A detailed in silico target prediction study was then performed to identify the genes regulated by DE microRNAs. We are currently validating the in silico analysis results by cell transfection with appropriate reporter vectors and microRNA mimics. The identification of targets for dysregulated microRNAs is expected to provide insights into transcriptional regulatory circuits subject to changes in perturbations of cholesterol metabolism.

Durante il mio periodo formativo come Dottoranda, ho contribuito alla realizzazione di differenti progetti di ricerca, tutti relativi allo studio dell’aterosclerosi e del metabolismo del colesterolo attraverso l’utilizzo di modelli animali. Tra questi, ho incentrato la mia tesi principalmente su due degli studi che mi vedono come co-autore. Ho inoltre riportato anche un terzo progetto, ancora in corso e i cui risultati preliminari sono stati presentati a congressi nazionali ed internazionali. Nello specifico, la tesi include lo studio dell’effetto di un idrolisato proteico di salmone sullo sviluppo di aterosclerosi in topi ApoE-deficienti, sulla base di precedenti evidenze illustranti come peptidi e idrolisati derivati dal pesce siano in grado di esercitare effetti benefici su fattori di rischio cardiovascolare. Topi alimentati per 12 settimane con una dieta ad alto contenuto di grassi con il 5% di idrolisato proteico di salmone hanno mostrato un’area di placca aterosclerotica significativamente ridotta a livello dell’arco e del seno aortico versus i topi controllo. L’effetto di questa supplementazione dietetica è stato dimostrato essere mediato dall’inibizione dell’attivazione dell’infiammazione sistemica e appare essere indipendente da cambiamenti nei livelli dei lipidi nel plasma. Inoltre, la presente tesi descrive lo studio dell’effetto della L-omoarginina, un omologo della L-arginina derivato dalla lisina, sull’iperplasia neointimale, una condizione patologica che può essere riscontrata a livello dei siti interessati da aterosclerosi subclinica o in seguito ad interventi di angioplastica. Dati emersi da studi clinici hanno evidenziato come la L-omoarginina possa essere considerata un nuovo biomarker per le patologie cardiovascolari. In particolare, due indipendenti studi di Genome-Wide Association hanno messo in luce una associazione tra le concentrazioni nel plasma di omoarginina e SNPs relativi al gene che codifica per l’arginina:glicina amidinotransferasi, un enzima che gioca un ruolo centrale nel metabolismo energetico, in quanto coinvolto nella sintesi dell’omoarginina e, indirettamente, della creatina. Per la prima volta, abbiamo dimostrato che il trattamento in vivo con omoarginina in un modello di balloon injury carotideo nel ratto è in grado di inibire la formazione di neointima, almeno in parte, inibendo la proliferazione delle cellule vascolari e incrementando sia la disponibilità di arginina che la produzione di ossido nitrico, suggerendo l’omoarginina come una potenziale opzione terapeutica per la protezione cardiovascolare. Infine, la presente tesi include la caratterizzazione, ancora in corso, dei pattern di espressione dei microRNA in differenti tessuti coinvolti nel metabolismo del colesterolo e nell’aterosclerosi raccolti da topi Ldlr-deficienti, Pcsk9-deficienti e wild-type C57BL/6 in seguito ad alimentazione con dieta standard o ad alto contenuto di grassi. Abbiamo identificato i microRNA che sono risultati differenzialmente espressi (DE) nelle nostre condizioni sperimentali attraverso procedure di sequenziamento ed analisi bioinformatiche ad hoc. E’ stata quindi effettuata una accurata analisi in silico di predizione del target al fine di identificare i geni regolati dai microRNA DE. Attualmente stiamo validando i risultati dell’analisi in silico attraverso esperimenti di trasfezione cellulare con vettori reporter appropriati e microRNA mimics. L’identificazione di bersagli per microRNA disregolati potrebbe quindi contribuire alla definizione di circuiti regolatori trascrizionali soggetti ad alterazioni in condizioni caratterizzate da perturbazioni del metabolismo del colesterolo.

(2017). ROLE OF TREATMENTS AND GENETIC ALTERATIONS ON ATHEROSCLEROSIS AND CHOLESTEROL METABOLISM: STUDIES IN ANIMAL MODELS. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2017).

ROLE OF TREATMENTS AND GENETIC ALTERATIONS ON ATHEROSCLEROSIS AND CHOLESTEROL METABOLISM: STUDIES IN ANIMAL MODELS

DELLERA, FEDERICA
2017

Abstract

During my educational path as a PhD Student, I have contributed to the realization of different research projects, all related to the study of atherosclerosis and cholesterol metabolism through the use of animal models. Among these, I focused my PhD thesis mainly on two of the studies I co-authored, one as first author. Moreover, a third study, still ongoing and whose results have been presented at National and International conferences, have been discussed. Specifically, my thesis includes a study of the effect of a salmon protein hydrolysate on atherosclerosis development in ApoE-deficient mice, on the basis of previous evidences indicating that fish protein peptides and hydrolysates are able to exert beneficial effects on cardiovascular risk factors. Mice fed a high-fat diet with 5% salmon protein hydrolysate for 12 weeks showed a significantly reduced atherosclerotic plaque area in the aortic arch and aortic sinus versus controls. The effect of this dietary supplementation was shown to be mediated by the inhibition of systemic inflammation activation and it seemed to be independent of changes in plasma lipid levels. Moreover, I described and discussed the investigation of the effect of L-homoarginine, an L-arginine homologue derived from lysine, on neointimal hyperplasia, a pathological condition which can occur at sites of subclinical atherosclerosis or after angioplasty interventions. Data from clinical studies indicated L-homoarginine as a novel biomarker for cardiovascular diseases. In particular, two independent Genome-Wide Association Studies identified an association between L-homoarginine plasma levels and SNPs related to the arginine:glycine amidinotransferase gene, which plays a central role in energy metabolism, being involved in homoarginine and, indirectly, creatine synthesis. For the first time, we have demonstrated that in vivo administration of homoarginine in a rat model of carotid balloon injury was able to inhibit neointimal formation, at least in part, by inhibiting vascular cell proliferation and by increasing both arginine availability and nitric oxide production, suggesting homoarginine as a potential therapeutical option for cardiovascular protection. Finally, my thesis also includes the ongoing characterization of microRNA expression patterns in tissues involved in cholesterol metabolism and atherosclerosis, harvested from Ldlr-deficient, Pcsk9-deficient and wild-type C57BL/6 mice after chow or a high fat feeding. We have identified the microRNAs which resulted differentially expressed (DE) in our experimental conditions by high-throughput sequencing and bioinformatics analyses. A detailed in silico target prediction study was then performed to identify the genes regulated by DE microRNAs. We are currently validating the in silico analysis results by cell transfection with appropriate reporter vectors and microRNA mimics. The identification of targets for dysregulated microRNAs is expected to provide insights into transcriptional regulatory circuits subject to changes in perturbations of cholesterol metabolism.
GIOVANNONI, ROBERTO
atherosclerosis,; cholesterol,; homoarginine,; salmon,; microRNA
atherosclerosis,; cholesterol,; homoarginine,; salmon,; microRNA
MED/04 - PATOLOGIA GENERALE
English
23-mar-2017
MEDICINA TRASLAZIONALE E MOLECOLARE - DIMET - 76R
29
2015/2016
open
(2017). ROLE OF TREATMENTS AND GENETIC ALTERATIONS ON ATHEROSCLEROSIS AND CHOLESTEROL METABOLISM: STUDIES IN ANIMAL MODELS. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2017).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/153228
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