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The fibrotic tissue and the stroma adjacent to cancer cells are characterised by the presence of activated fibroblasts (myofibroblasts) which play a role in creating a supportive tissue charac-terised by abundant extracellular matrix (ECM) secretion. The myofibroblasts remodel this tissue through secreted molecules and modulation of their cytoskeleton and specialized con-tractile structures. The non-receptor protein tyrosine kinase Arg (also called Abl2) has the unique ability to bind directly to the actin cytoskeleton, transducing diverse extracellular sig-nals into cytoskeletal rearrangements. In this study we analysed the 1ALCTL and 1BLCTL Arg isoforms in Arg-/- murine embryonal fibroblasts (MEF) cell line, focusing on their capac-ity to activate fibroblasts and to remodel ECM. The results obtained showed that Arg isoform 1BLCTL has a major role in proliferation, migration/invasion of MEF and in inducing a milieu able to modulate tumour cell morphology, while 1ALCTL isoform has a role in MEF adhesion maintaining active focal adhesions. On the whole, the presence of Arg in MEF supports the proliferation, activa-tion, adhesion, ECM contraction and stiffness, while the absence of Arg affected these myofibroblast features

Background: Atherosclerosis is a slowly progressing, chronic multifactorial disease characterized by the accumulation of lipids, inflammatory cells, and fibrous tissue that drives to the formation of asymmetric focal thickenings in the tunica intima of large and mid-sized arteries. Despite the high therapeutic potential of ApoA-1 proteins, the purification and delivery into the disordered organisms of these drugs is still limited by low efficiency in these processes. Methods and results: We report here a novel production and delivery system of anti-atherogenic APOA-1Milano muteins (APOA-1M) by means of genetically modified rice plants. APOA-1M, delivered as protein extracts from transgenic rice seeds, significantly reduced macrophage activation and foam cell formation in vitro in oxLDL-loaded THP-1 model. The APOA-1M delivery method and therapeutic efficacy was tested in healthy mice and in Apoe −/− mice fed with high cholesterol diet (Western Diet, WD). APOA-1M rice milk significantly reduced atherosclerotic plaque size and lipids composition in aortic sinus and aortic arch of WD-fed Apoe −/− mice as compared to wild type rice milk-treated, WD-fed Apoe −/− mice. APOA-1M rice milk also significantly reduced macrophage number in liver of WD-fed Apoe −/− mice as compared to WT rice milk treated mice. Translational impact: The delivery of therapeutic APOA-1M full length proteins via oral administration of rice seeds protein extracts (the ‘rice milk’) to the disordered organism, without any need of purification, might overcome the main APOA1-based therapies’ limitations and improve the use of this molecules as therapeutic agents for cardiovascular patients.

Cancer-initiating cells (CICs) have been implicated in tumor development and aggressiveness. In ovarian carcinoma (OC), CICs drive tumor formation, dissemination, and recurrence, as well as drug resistance, thus accounting for the high death-to-incidence ratio of this neoplasm. However, the molecular mechanisms that underlie such a pathogenic role of ovarian CICs (OCICs) remain elusive. Here, we have capitalized on primary cells either from OC or from its tissues of origin to obtain the transcriptomic profile associated with OCICs. Among the genes differentially expressed in OCICs, we focused on CD73, which encodes the membrane-associated 5′-ectonucleotidase. The genetic inactivation of CD73 in OC cells revealed that this molecule is causally involved in sphere formation and tumor initiation, thus emerging as a driver of OCIC function. Furthermore, functional inhibition of CD73 via either a chemical compound or a neutralizing antibody reduced sphere formation and tumorigenesis, highlighting the druggability of CD73 in the context of OCIC-directed therapies. The biological function of CD73 in OCICs required its enzymatic activity and involved adenosine signaling. Mechanistically, CD73 promotes the expression of stemness and epithelial-mesenchymal transition-associated genes, implying a regulation of OCIC function at the transcriptional level. CD73, therefore, is involved in OCIC biology and may represent a therapeutic target for innovative treatments aimed at OC eradication. Cavallaro et al. characterized the transcriptome of OCIC-enriched primary cultures and found CD73 as an upregulated gene. CD73 was then shown to regulate the expression of stemness and EMT-associated genes. The expression and function of CD73 in OCICs is required for tumor initiation, and CD73-targeted drugs decrease the rate of tumor take and inhibit cancer growth.

Inwardly rectifying potassium channels (Kir) have been historically associated to several cardiovascular disorders. In particular, loss-of-function mutations in the Kir2.1 channel have been reported in cases affected by Andersen-Tawil syndrome while gain-of-function mutations in the same channel cause the short QT3 syndrome. Recently, a missense mutation in Kir2.1, as well as mutations in the Kir4.1, were reported to be involved in autism spectrum disorders (ASDs) suggesting a role of potassium channels in these diseases and introducing the idea of the existence of K+ channel ASDs. Here, we report the identification in an Italian affected family of a novel missense mutation (p.Phe58Ser) in the KCNJ2 gene detected in heterozygosity in a proband affected by autism and borderline for short QT syndrome type 3. The mutation is located in the N-terminal region of the gene coding for the Kir2.1 channel and in particular in a very conserved domain. In vitro assays demonstrated that this mutation results in an increase of the channel conductance and in its open probability. This gain-of-function of the protein is consistent with the autistic phenotype, which is normally associated to an altered neuronal excitability.

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, 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 fac

Background. The management of modifiable risk factors exposure, in particular dyslipidemia, is the first line of intervention in preventing cardiovascular events. HDLs have been demonstrated to have anti-inflammatory and atheroprotective properties and, among HDLs, Apolipoprotein A-I (APOA-1), which promotes reverse cholesterol efflux, has been deeply investigated for the great therapeutic potential, particularly emphasized for the naturally occurring mutation APOA-1Milano (APOA-1M). Despite the high therapeutic potential of these molecules, their purification and delivery into the disordered organism is still limited by a very low efficiency in these processes. Aim. To develop an efficient system of production and delivery of APOA-1 muteins without need of purification. Methods and Results. Rice plants were genetically modified to express APOA-1M protein in their seeds. Protein extract from transgenic rice seeds (the ‘APOA-1M rice milk’) was tested for functionality in vitro on THP-1 macrophages exposed to oxLDL. Protein extract from wild type rice seeds (the ‘WT rice milk’) was used as control. The APOA-1M rice milk, but not the WT rice milk, significantly reduced expression of MCP-1 in oxLDL-loaded THP-1 macrophages in a dose-dependent manner and it promoted reverse cholesterol efflux in THP-1 macrophages. The lack of toxicity and the tolerability of the orally administered APOA-1M rice milk was evaluated in healthy mice. In an early-intermediate atherosclerotic mouse model (Apoe-/-mice fed with Western Diet for 8 weeks), 3 weeks of APOA-1M, but not the WT, rice milk treatment (15d, 5d/week, by oral gavage) significantly reduced area of lipid deposition and lipids concentration at aortic arch (en face analysis). Moreover, the APOA-1M, but not the WT, rice milk treatment reduced the hepatic CD68-positive cells and ameliorated the lipid management gene expression profile in liver of WD-fed Apoe-/-mice. Interestingly, all these findings were observed in mice still e

Ischemia-reperfusion injury (IRI) and oxidative stress still limit the survival of cells and organs in xenotransplantation models. Ectonucleotidases play an important role in inflammation and IRI in transplantation settings. We tested the potential protective effects derived by the co-expression of the two main vascular ectonucleotidases, ecto-5’-nucleotidase (E5NT) and ecto nucleoside triphosphate diphosphohydrolase 1 (ENTPD1), in an in vitro model of H2O2-induced oxidative stress and cytotoxicity. We produced a dicistronic plasmid (named pCX-DI-2A) for the co-expression of human E5NT and ENTPD1 by using the F2A technology. pCX-DI-2A-transfected porcine endothelial cells simultaneously overexpressed hE5NT and hENTPD1, which were correctly processed and localized on the plasma membrane. Furthermore, such co-expression system led to the synergistic enzymatic activity of hE5NT and hENTPD1 as shown by the efficient catabolism of pro-inflammatory and pro-thrombotic extracellular adenine nucleotides along with the enhanced production of the anti-inflammatory molecule adenosine. Interestingly, we found that the hE5NT/hENTPD1 co-expression system conferred protection to cells against H2O2-induced oxidative stress and cytotoxicity. pCX-DI-2A-transfected cells showed reduced activation of caspase 3/7 and cytotoxicity than mock-, hE5NT- and hENTPD1-transfected cells. Furthermore, pCX-DI-2A-transfected cells showed decreased H2O2-induced production of ROS as compared to the other control cell lines. 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, extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). Our data add new insights to the protective effects of the combination of hE5NT and hENTPD1 against oxidative stress and constitute a proof of concept for testing this new genetic com

We developed an F2A-based multicistronic system to evaluate functional effects of co-expression of three proteins important for xenotransplantation: heme oxygenase 1 (HO1), ecto-5'-nucleotidase (E5NT) and ecto-nucleoside triphosphate diphosphohydrolase-1 (ENTPD1). The tricistronic p2A plasmid that we constructed was able to efficiently drive concurrent expression of HO1, E5NT and ENTPD1 in HEK293T cells. All three overexpressed proteins possessed relevant enzymatic activities, while addition of furin site interfered with protein expression and activity. We conclude that our tricistronic p2A construct is effective and optimal to test the combined protective effects of HO1, E5NT and ENTPD1 against xeno-rejection mechanisms.