Oxidative stress is a major and recurring cause of damage during inflammation, as following ischemia-reperfusion injury (IRI). Increasing evidences report purinergic signaling as a key pathway in inflammation and IRI settings. Indeed, following IRI, extracellular ATP and ADP accumulate in the sites of injury leading to pro-inflammatory and pro-thrombotic responses on vascular and immune cells. The extracellular levels of adenine nucleotides are mainly modulated by the ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) and ecto-5-nucleotidase (E5NT), whose combined enzymatic activities convert ATP into adenosine, an anti-inflammatory molecule. On the other hand, heme oxygenase-1 (HO-1) gained much attention in the context of IRI and transplantation because of its role in down-regulating inflammatory response and apoptosis and the well-known vasodilatory, anti-oxidant, and anti-inflammatory properties of its catabolites, carbon monoxide and bilirubin. Several biomedical applications, such as xenotransplantation, require multiple genetic modifications in eukaryotic cells to ensure a successful outcome. Advances in genetic engineering technologies have led to the development of efficient polycistronic vectors based on the use of the 2A self-processing oligopeptide. We developed an F2A-based multicistronic system to evaluate functional effects of co-expression of the three anti-inflammatory proteins, HO-1, E5NT and ENTPD1. The novel tricistronic p2A plasmid we designed and produced was able to efficiently drive simultaneous expression of the three genes in HEK293T cells. All the exogenously overexpressed proteins possessed relevant enzymatic activities and the tricistronic p2A construct resulted to be effective and optimal to test the combined protective effects of HO1, E5NT and ENTPD1 against inflammation and oxidative stress injury. Thus, we evaluated the potential protection mediated by this new combination of genes in NIH3T3 cells exposed to tumor necrosis factor alpha (TNF-α), as a key pro-inflammatory cytokine involved in the inflammatory response during IRI. NIH3T3 cells stably transfected with an updated tricistronic plasmid (pCX-TRI-2A) showed a synergic cytoprotective effect against TNF-α-induced injury and cell death and this protection may be at least in part explained by the induction of a pro-survival phenotype in these cells. Since encouraging results were obtained in our in vitro model, the tricistronic cassette was used for the production of a multi-gene transgenic mouse model for the over-expression of hHO1, hE5NT and hENTPD1. Three transgenic mouse strains were produced and the proper enzymatic activity of the two ectonucleotidases was observed in different tissues of the mouse strains. We furthermore investigated whether the co-expression of the two main vascular ectonucleotidases, might protect against H2O2-induced oxidative stress in vitro. To this purpose, we assembled a dicistronic plasmid (pCX-DI-2A) for the co-expression of hE5NT and hENTPD1. pCX-DI-2A-transfected porcine iliac endothelial cells (PIEC) simultaneously overexpressed and correctly processed the two human proteins and such co-expression system led to the synergistic enzymatic activity of hE5NT and hENTPD1 and a concomitant reduction of H2O2-induced ROS production, cytotoxicity and apoptosis. Interestingly, we showed that the cytoprotective phenotype observed in pCX-DI-2A-transfected cells was associated with higher detoxifying activity of catalase as well as increased activation of the survival signaling molecules Akt, ERK1/2 and p38 MAPKs. Overall, our work add new insights to the understanding of the anti-inflammatory effect and the ability to counteract oxidative stress mediated by a novel combination of the three human genes and constitute a proof of concept for testing this new genetic combination in pre-clinical models relevant for the study of IRI and inflammatory-based diseases, as transplantation and atherosclerosis.
Lo stress ossidativo è una causa ricorrente di danno durante l’infiammazione associata al danno da ischemia-riperfusione (I/R). E’ noto che il pathway purinergico abbia un ruolo chiave nel contesto di I/R. Durante il processo di I/R, infatti, l'ATP e l'ADP extracellulari si accumulano nei siti di lesione promuovendo risposte pro-infiammatorie e pro-trombotiche in cellule vascolari e immunitarie. I livelli extracellulari dei nucleotidi adeninici sono principalmente modulati dall'ectonucleoside trifosfato difosfidridrasi 1 (ENTPD1) e dall'ecto-5-nucleotidasi (E5NT), le cui attività enzimatiche combinate trasformano l'ATP in adenosina, una molecola anti-infiammatoria. Inoltre, l'eme ossigenasi-1 (HO-1) ha acquisito molta attenzione nel contesto dell'I/R e del trapianto grazie al suo ruolo nel contrastare la risposta infiammatoria e l'apoptosi e agli effetti vasodilatatori, antiossidanti e anti-infiammatori mediati dai suoi cataboliti, monossido di carbonio e bilirubina. Diverse applicazioni biomediche, ad esempio lo xenotrapianto, richiedono molteplici modificazioni genetiche dei sistemi eucariotici per garantire successo come applicazioni cliniche. I progressi nelle tecnologie di ingegneria genetica hanno portato allo sviluppo di efficienti vettori policistronici basati sull'utilizzo del peptide virale 2A. Il nostro gruppo ha sviluppato un sistema multicistronico basato sul peptide F2A per valutare gli effetti funzionali della co-espressione delle tre proteine anti-infiammatorie, HO-1, E5NT e ENTPD1. Il plasmide tricistronico p2A è in grado di guidare efficientemente l'espressione simultanea dei tre geni nelle cellule HEK293T. Inoltre, tali proteine esogene hanno mostrato attività enzimatiche rilevanti, rendendo il costrutto tricistronico uno strumento efficace e ottimale per testare gli effetti protettivi combinati di HO1, E5NT e ENTPD1 contro l'infiammazione e le lesioni da stress ossidativo. Pertanto, abbiamo valutato la potenziale protezione mediata da questa nuova combinazione di geni nelle cellule NIH3T3 esposte a TNF-α, una citochina pro-infiammatoria coinvolta nella risposta infiammatoria durante l'I/R. Le cellule NIH3T3 transfettate stabilmente con una versione aggiornata del plasmide tricistronico (pCX-TRI-2A) hanno mostrato un effetto citoprotettivo sinergico contro il danno e la morte cellulare indotti da TNF-α e questa protezione può essere almeno in parte spiegata dall'induzione di un fenotipo di sopravvivenza in queste cellule. Poiché sono stati ottenuti risultati incoraggianti nel nostro modello in vitro, la cassetta tricistronica è stata utilizzata per la produzione di un modello di topo multi-transgenico. Sono state prodotte tre linee transgeniche che esprimono le due ectonucleotidasi enzimaticamente attive in diversi tessuti. Abbiamo infine valutato se la co-espressione delle due ectonucleotidasi vascolari, protegga dallo stress ossidativo indotto da H2O2 in vitro. A tale scopo, abbiamo assemblato un plasmide dicistronico (pCX-DI-2A) e trasfettato stabilmente cellule endoteliali iliache porcine (PIEC). Tali cellule esprimono simultaneamente le due proteine che vengono processate correttamente e presentano un’azione sinergica che comporta una riduzione della produzione di ROS, citotossicità e apoptosi indotte da H2O2. È interessante notare che il fenotipo citoprotettivo osservato nelle cellule trasfettate con pCX-DI-2A è associato ad una maggiore attività detossificante della catalasi e ad una maggiore attivazione delle molecole dei signaling pathways Akt, ERK1/2 e p38 MAPK. Nel complesso, il nostro lavoro aggiunge nuove conoscenze alla comprensione dell'effetto anti-infiammatorio e anti-ossidativo mediato da una nuova combinazione di tre geni umani e costituisce una dimostrazione preliminare per poter testare questa nuova combinazione in modelli pre-clinici rilevanti per lo studio dell'I/R e delle malattie su base infiammatoria, come trapianto e aterosclerosi.
(2018). Investigating cytoprotective molecular mechanisms against inflammation and oxidative stress stimuli by using newly developed multi-cistronic genetic tools. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2018).
Investigating cytoprotective molecular mechanisms against inflammation and oxidative stress stimuli by using newly developed multi-cistronic genetic tools
CHISCI, ELISA
2018
Abstract
Oxidative stress is a major and recurring cause of damage during inflammation, as following ischemia-reperfusion injury (IRI). Increasing evidences report purinergic signaling as a key pathway in inflammation and IRI settings. Indeed, following IRI, extracellular ATP and ADP accumulate in the sites of injury leading to pro-inflammatory and pro-thrombotic responses on vascular and immune cells. The extracellular levels of adenine nucleotides are mainly modulated by the ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) and ecto-5-nucleotidase (E5NT), whose combined enzymatic activities convert ATP into adenosine, an anti-inflammatory molecule. On the other hand, heme oxygenase-1 (HO-1) gained much attention in the context of IRI and transplantation because of its role in down-regulating inflammatory response and apoptosis and the well-known vasodilatory, anti-oxidant, and anti-inflammatory properties of its catabolites, carbon monoxide and bilirubin. Several biomedical applications, such as xenotransplantation, require multiple genetic modifications in eukaryotic cells to ensure a successful outcome. Advances in genetic engineering technologies have led to the development of efficient polycistronic vectors based on the use of the 2A self-processing oligopeptide. We developed an F2A-based multicistronic system to evaluate functional effects of co-expression of the three anti-inflammatory proteins, HO-1, E5NT and ENTPD1. The novel tricistronic p2A plasmid we designed and produced was able to efficiently drive simultaneous expression of the three genes in HEK293T cells. All the exogenously overexpressed proteins possessed relevant enzymatic activities and the tricistronic p2A construct resulted to be effective and optimal to test the combined protective effects of HO1, E5NT and ENTPD1 against inflammation and oxidative stress injury. Thus, we evaluated the potential protection mediated by this new combination of genes in NIH3T3 cells exposed to tumor necrosis factor alpha (TNF-α), as a key pro-inflammatory cytokine involved in the inflammatory response during IRI. NIH3T3 cells stably transfected with an updated tricistronic plasmid (pCX-TRI-2A) showed a synergic cytoprotective effect against TNF-α-induced injury and cell death and this protection may be at least in part explained by the induction of a pro-survival phenotype in these cells. Since encouraging results were obtained in our in vitro model, the tricistronic cassette was used for the production of a multi-gene transgenic mouse model for the over-expression of hHO1, hE5NT and hENTPD1. Three transgenic mouse strains were produced and the proper enzymatic activity of the two ectonucleotidases was observed in different tissues of the mouse strains. We furthermore investigated whether the co-expression of the two main vascular ectonucleotidases, might protect against H2O2-induced oxidative stress in vitro. To this purpose, we assembled a dicistronic plasmid (pCX-DI-2A) for the co-expression of hE5NT and hENTPD1. pCX-DI-2A-transfected porcine iliac endothelial cells (PIEC) simultaneously overexpressed and correctly processed the two human proteins and such co-expression system led to the synergistic enzymatic activity of hE5NT and hENTPD1 and a concomitant reduction of H2O2-induced ROS production, cytotoxicity and apoptosis. Interestingly, we showed that the cytoprotective phenotype observed in pCX-DI-2A-transfected cells was associated with higher detoxifying activity of catalase as well as increased activation of the survival signaling molecules Akt, ERK1/2 and p38 MAPKs. Overall, our work add new insights to the understanding of the anti-inflammatory effect and the ability to counteract oxidative stress mediated by a novel combination of the three human genes and constitute a proof of concept for testing this new genetic combination in pre-clinical models relevant for the study of IRI and inflammatory-based diseases, as transplantation and atherosclerosis.File | Dimensione | Formato | |
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phd_unimib_718273.pdf
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Descrizione: tesi di dottorato
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Doctoral thesis
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