Antitumoral effects of pharmacological ascorbate on gastric cancer cells: GLUT1 expression may not tell the whole story

The recently reported results of Lu et al. (Theranostics. 2018; 8: 1312-26) – highlighting GLUT1 expression as a marker for sensitivity of gastric cancer cells to therapeutic doses of ascorbate – are discussed in the light of additional factors potentially affecting the underlying processes, such as the concomitant expression of membrane gamma-glutamyltransferase activity, the resistance of cancer cells to oxidative injury and other known biomarkers

Monocytes/macrophages activation contributes to b-gamma-glutamyltransferase accumulation inside atherosclerotic plaques

Gamma-glutamyltransferase (GGT) is a well-established independent risk factor for cardiovascular mortality related to atherosclerotic disease. Four GGT fractions have been identified in plasma, but only b-GGT fraction accumulates in atherosclerotic plaques, and correlates with other histological markers of vulnerability. The present study was aimed to evaluate whether macrophagic lineage cells may provide a source of b-GGT within the atherosclerotic plaque

p53 Activation Effect in the Balance of T Regulatory and Effector Cell Subsets in Patients With Thyroid Cancer and Autoimmunity

Carcinomas evade the host immune system by negatively modulating CD4+ and CD8+ T effector lymphocytes through forkhead box protein 3 (FOXP3) positive T regulatory cells' increased activity. Furthermore, interaction of the programmed cell death 1 (PD1) molecule and its ligand programmed cell death ligand 1 (PDL1) inhibits the antitumor activity of PD1+ T lymphocytes. Immunotherapy has become a powerful strategy for tailored cancer patients' treatment both in adult and pediatric patients aiming to generate potent antitumor responses. Nevertheless, immunotherapies can generate autoimmune responses. This study aimed to investigate the potential effect of the transformation-related protein 53 (p53) reactivation by a peptide-based inhibitor of the MDM2/MDM4 heterodimer (Pep3) on the immune response in a solid cancer, i.e., thyroid carcinoma frequently presenting with thyroid autoimmunity. In peripheral blood mononuclear cell of thyroid cancer patients, Pep3 treatment alters percentages of CD8+ and CD4+ T regulatory and CD8+ and CD4+ T effector cells and favors an anticancer immune response. Of note that reduced frequencies of activated CD8+ and CD4+ T effector cells do not support autoimmunity progression. In evaluating PD1 expression under p53 activation, a significant decrease of activated CD4+PD1+ cells was detected in thyroid cancer patients, suggesting a defective regulation in the initial activation stage, therefore generating a protective condition toward autoimmune progression

Angiotensin {II}-induced redox-sensitive {SGLT}1 and 2 expression promotes high glucose-induced endothelial cell senescence

High glucose (HG)-induced endothelial senescence and dysfunction contribute to the increased cardiovascular risk in diabetes. Empagliflozin, a selective sodium glucose co-transporter2 (SGLT2) inhibitor, reduced the risk of cardiovascular mortality in type 2 diabetic patients but the protective mechanism remains unclear. This study examines the role of SGLT2 in HG-induced endothelial senescence and dysfunction. Porcine coronary artery cultured endothelial cells (ECs) or segments were exposed to HG (25 mmol/L) before determination of senescence-associated beta-galactosidase activity, protein level by Western blot and immunofluorescence staining, mRNA by RT-PCR, nitric oxide (NO) by electron paramagnetic resonance, oxidative stress using dihydroethidium and glucose uptake using 2-NBD-glucose. HG increased ECs senescence markers and oxidative stress, down-regulated eNOS expression and NO formation, and induced the expression of VCAM-1, tissue factor, and the local angiotensin system, all these effects were prevented by empagliflozin. Empagliflozin and LX-4211 (dual SGLT1/2 inhibitor) reduced glucose uptake stimulated by HG and H2O2 in ECs. HG increased SGLT1 and 2 protein levels in cultured ECs and native endothelium. Inhibition of the angiotensin system prevented HG-induced ECs senescence and SGLT1 and 2 expression. Thus, HG-induced ECs ageing is driven by the local angiotensin system via the redox-sensitive up-regulation of SGLT1 and 2, and, in turn, enhanced glucotoxicity

Inflammation et stress oxydant dans l'athérosclérose : rôle dans les réponses vasculaires des S-nitrosothiols

Atherosclerosis is a slowly progressing chronic disease characterized by the formation of atherosclerotic plaques consisting of accumulated low density lipoprotein (LDL), leukocytes, foam cells, migrated smooth muscle cells (SMCs) and altered endothelial cells (ECs), leading to the formation of necrotic cores with calcified regions. Atherosclerosis genesis and subsequent instability of atherosclerotic plaques result from a synergy between inflammation and oxidative stress. Current data identified several macrophage populations within the atherosclerotic plaque showing different inflammatory phenotypes (pro-inflammatory: M1, anti-inflammatory: M2) or functions in response to redox changes in the environment (Mox). The oxidative stress linked to inflammation plays an important role in (i) endothelial dysfunction, with reduced nitric oxide (NO) bioavailability, (ii) LDL oxidation, (iii) lesion remodeling (regulation of proteases and antiproteases) and (iv) SMCs proliferation. Indeed, SMCs are the second more abundant cell type, after macrophages, in the atherosclerotic lesion because their dedifferentiation from contractile to secreting phenotype increased their proliferation and migration capacity. NO donors, like S-nitrosothiols, also known to protect from oxidative stress by S-nitrosation, could counteract this NO deficiency. Among them, the S-nitrosoglutathione (GSNO), a physiological storage form of NO in tissues, specifically catabolized by the gamma-glutamyltransferase (GGT) is considered. Recently, it has been shown that the increased serum level of GGT is an independent risk factor for cardiovascular mortality related to atherosclerotic disease. In particular, only the big fraction (b-GGT) has been detected inside human atherosclerotic plaques associated to CD68+ macrophage-derived foam cells. As macrophages and SMCs are the main cell types found in atherosclerotic lesion and seemed to be colocalized with GGT, the attention of this thesis work was focused on the understanding of GGT provenance and its role in the GSNO metabolism within the atherosclerotic plaque. A first part of the thesis was to identify the origin of GGT accumulating inside atherosclerotic plaques, and to decipher between inflammation and oxidative stress stimuli, which one is responsible of GGT accumulation in atherosclerotic plaques. The second part was dedicated to the restoration of NO bioavailability within SMCs under oxidative stress with a focus on the identification of S-nitrosated proteinsL'athérosclérose est une maladie chronique à évolution lente caractérisée par la formation de plaques d'athérome, consistant en l’accumulation de lipoprotéines de basse densité (LDL), de leucocytes, de cellules spumeuses, la migration des cellules musculaires lisses (CML) et l’altération des cellules endotheliales (ECs). Ces phénomènes conduisent à la formation d'un noyau nécrotique incluant des régions calcifiées. La genèse de l'athérosclérose et de l’instabilité de la plaque d’athérome sont le résultat d'une synergie entre inflammation et stress oxydant. Les données actuelles identifient plusieurs populations de macrophages dans la plaque d’athérome présentant différents phénotypes en lien avec l’inflammation (pro-inflammatoire: M1, anti-inflammatoire: M2) ou avec des modifications redox de l’environnement (Mox). Stress oxydant et inflammation sont liés et jouent un rôle important dans (i) la dysfonction endothéliale induisant une diminution de la biodisponibilité du monoxyde d’azote (NO), (ii) l'oxydation des LDL, (iii) le remodelage de la lésion (régulation de protéases et d’antiprotéases) et (iv) la prolifération des CML. Les CML sont le deuxième type cellulaire le plus abondant dans la lésion athérosclérotique après les macrophages, leur hyperprolifération est la conséquence d’une dédifférenciation cellulaire d’un phénotype contractile à sécrétoire, augmentant leur capacité proliférative et migratoire. Les donneurs de NO, comme les S-nitrosothiols, connus également pour protéger contre le stress oxydant grâce essentiellement à la S-nitrosation, peuvent contrer la carence en NO. Parmi eux, le S- nitrosoglutathion (GSNO), forme physiologique de stockage de NO dans les tissus, spécifiquement metabolisé par la gamma-glutamyl transférase (GGT) peut être envisagé. La corrélation entre l’augmentation des concentrations sériques de GGT et les facteurs de risque cardiovasculaire a récemment été démontrée. En particulier, seule la b-GGT s'accumule dans les plaques d'athérome, et concorde avec l’apparition d'autres marqueurs histologiques de vulnérabilité de la plaque. Étant donné que, les macrophages et les CML sont les principaux types cellulaires retrouvés dans les lésions athérosclérotiques et semblaient être colocalisés avec la GGT, l'attention de ce travail de thèse a été centrée sur la compréhension de la provenance de la GGT et son rôle dans le métabolisme du GSNO au sein de la plaque d’athérome. Une première partie de ce manuscrit vise à identifier l'origine de la GGT accumulée dans la plaque d’athérome, et à élucider entre le stimulus inflammatoire et oxydant, qui est responsable de l'accumulation de GGT dans la plaque d'athérome. La deuxième partie a été consacrée à la restauration de la biodisponibilité de NO dans les CML en condition de stress oxydant avec un intérêt particulier porté sur l'identification des protéines S-nitrosé

Inflammation and oxidative stress in atherosclerosis : role of S-nitrosothiols in the vascular responses

L'athérosclérose est une maladie chronique à évolution lente caractérisée par la formation de plaques d'athérome, consistant en l’accumulation de lipoprotéines de basse densité (LDL), de leucocytes, de cellules spumeuses, la migration des cellules musculaires lisses (CML) et l’altération des cellules endotheliales (ECs). Ces phénomènes conduisent à la formation d'un noyau nécrotique incluant des régions calcifiées. La genèse de l'athérosclérose et de l’instabilité de la plaque d’athérome sont le résultat d'une synergie entre inflammation et stress oxydant. Les données actuelles identifient plusieurs populations de macrophages dans la plaque d’athérome présentant différents phénotypes en lien avec l’inflammation (pro-inflammatoire: M1, anti-inflammatoire: M2) ou avec des modifications redox de l’environnement (Mox). Stress oxydant et inflammation sont liés et jouent un rôle important dans (i) la dysfonction endothéliale induisant une diminution de la biodisponibilité du monoxyde d’azote (NO), (ii) l'oxydation des LDL, (iii) le remodelage de la lésion (régulation de protéases et d’antiprotéases) et (iv) la prolifération des CML. Les CML sont le deuxième type cellulaire le plus abondant dans la lésion athérosclérotique après les macrophages, leur hyperprolifération est la conséquence d’une dédifférenciation cellulaire d’un phénotype contractile à sécrétoire, augmentant leur capacité proliférative et migratoire. Les donneurs de NO, comme les S-nitrosothiols, connus également pour protéger contre le stress oxydant grâce essentiellement à la S-nitrosation, peuvent contrer la carence en NO. Parmi eux, le S- nitrosoglutathion (GSNO), forme physiologique de stockage de NO dans les tissus, spécifiquement metabolisé par la gamma-glutamyl transférase (GGT) peut être envisagé. La corrélation entre l’augmentation des concentrations sériques de GGT et les facteurs de risque cardiovasculaire a récemment été démontrée. En particulier, seule la b-GGT s'accumule dans les plaques d'athérome, et concorde avec l’apparition d'autres marqueurs histologiques de vulnérabilité de la plaque. Étant donné que, les macrophages et les CML sont les principaux types cellulaires retrouvés dans les lésions athérosclérotiques et semblaient être colocalisés avec la GGT, l'attention de ce travail de thèse a été centrée sur la compréhension de la provenance de la GGT et son rôle dans le métabolisme du GSNO au sein de la plaque d’athérome. Une première partie de ce manuscrit vise à identifier l'origine de la GGT accumulée dans la plaque d’athérome, et à élucider entre le stimulus inflammatoire et oxydant, qui est responsable de l'accumulation de GGT dans la plaque d'athérome. La deuxième partie a été consacrée à la restauration de la biodisponibilité de NO dans les CML en condition de stress oxydant avec un intérêt particulier porté sur l'identification des protéines S-nitrosésAtherosclerosis is a slowly progressing chronic disease characterized by the formation of atherosclerotic plaques consisting of accumulated low density lipoprotein (LDL), leukocytes, foam cells, migrated smooth muscle cells (SMCs) and altered endothelial cells (ECs), leading to the formation of necrotic cores with calcified regions. Atherosclerosis genesis and subsequent instability of atherosclerotic plaques result from a synergy between inflammation and oxidative stress. Current data identified several macrophage populations within the atherosclerotic plaque showing different inflammatory phenotypes (pro-inflammatory: M1, anti-inflammatory: M2) or functions in response to redox changes in the environment (Mox). The oxidative stress linked to inflammation plays an important role in (i) endothelial dysfunction, with reduced nitric oxide (NO) bioavailability, (ii) LDL oxidation, (iii) lesion remodeling (regulation of proteases and antiproteases) and (iv) SMCs proliferation. Indeed, SMCs are the second more abundant cell type, after macrophages, in the atherosclerotic lesion because their dedifferentiation from contractile to secreting phenotype increased their proliferation and migration capacity. NO donors, like S-nitrosothiols, also known to protect from oxidative stress by S-nitrosation, could counteract this NO deficiency. Among them, the S-nitrosoglutathione (GSNO), a physiological storage form of NO in tissues, specifically catabolized by the gamma-glutamyltransferase (GGT) is considered. Recently, it has been shown that the increased serum level of GGT is an independent risk factor for cardiovascular mortality related to atherosclerotic disease. In particular, only the big fraction (b-GGT) has been detected inside human atherosclerotic plaques associated to CD68+ macrophage-derived foam cells. As macrophages and SMCs are the main cell types found in atherosclerotic lesion and seemed to be colocalized with GGT, the attention of this thesis work was focused on the understanding of GGT provenance and its role in the GSNO metabolism within the atherosclerotic plaque. A first part of the thesis was to identify the origin of GGT accumulating inside atherosclerotic plaques, and to decipher between inflammation and oxidative stress stimuli, which one is responsible of GGT accumulation in atherosclerotic plaques. The second part was dedicated to the restoration of NO bioavailability within SMCs under oxidative stress with a focus on the identification of S-nitrosated protein

Alterazioni trascrizionali del sistema dei peptidi natriuretici e dell'endotelina causate dall'obesita: studio nel tessuto cardiaco di ratti Zucker.

Introduzione: L’obesità è una malattia cronica multifattoriale e complessa in cui sono implicati sia fattori ambientali che genetici, è associata alla sindrome metabolica e rappresenta un fattore di rischio indipendente per varie patologie tra cui quelle cardiovascolari. Il sistema dei peptidi natriuretici (PN) può giocare un ruolo importante nella regolazione di questo processo, è stato infatti ipotizzato che negli obesi ci sia una ridotta risposta da parte di questi peptidi. D’altro canto una maggiore attività del sistema dell’endotelina (ET)-1 è stata associata allo sviluppo e progressione di numerose patologie cardiovascolari legate all’obesità. Lo scopo del lavoro di tesi è stato quello di valutare le variazioni di espressione a livello di mRNA del sistema dei PN contemporaneamente a quelle del sistema dell’ET-1, nel tessuto cardiaco di ratti Zucker di controllo (CO=20) e obesi (O=20) in modo da valutare le possibili alterazioni trascrizionali causate dall’obesità. È stata inoltre analizzata l’espressione a livello di mRNA cardiaco di IL-6 e TNF-α per valutare il possibile legame tra PN, sistema dell’ET-1 e processo infiammatorio. Infine, vista l'interazione nota tra l’asse tiroideo e il peso corporeo, sono stati determinati i livelli plasmatici degli ormoni tiroidei T3, T4 e TSH, sia in CO che in O. Materiali e Metodi: Il tessuto cardiaco è stato ottenuto da ratti maschi Zucker di 11-13 settimane di età, obesi (O=20, peso corporeo=374±8,5 g) e controlli (CO=20, peso corporeo=281±10 g). Gli animali sono stati studiati in condizione di digiuno (COdig, n=10, glicemia= 101,3±15,3 mg/dl; Odig, n=10, glicemia= 299,1±44,6 mg/dl) e durante induzione di iperglicemia in seguito ad iniezione di un bolo di glucosio (COiper, n=10, glicemia=324,4±22 mg/dl; Oiper, n=10, glicemia= 397,6±46,1 mg/dl). L’mRNA è stato estratto da campioni di tessuto cardiaco mediante il metodo della guanidina tiocianato-fenolo cloroformio e l’espressione cardiaca dell’mRNA codificante per i geni in esame è stata determinata con l’uso di Real Time-PCR (RT-PCR) dopo opportuna messa a punto del protocollo. Al fine di ottenere una corretta normalizzazione dei dati ottenuti dagli esperimenti di RT-PCR, l’analisi statistica è stata effettuata previa normalizzazione dei risultati con un set di geni di riferimento (SDHA, TBP, HPRT-1) specificatamente selezionati. Per determinare i livelli plasmatici degli ormoni tiroidei T3, T4 e TSH, sia in CO che in O, è stato utilizzato un sistema immunometrico multianalita, MagPix MILLIPORE. Risultati: Non sono state osservate differenze significative nell’espressione dei livelli di mRNA dell’ANP e del CNP mentre i livelli di espressione del BNP nel ratto obeso sono risultati significativamente diminuiti rispetto ai ratti di controllo (p=0,01). I ratti obesi rispetto ai ratti di controllo hanno evidenziato una down-regulation dei sottotipi recettoriali NPR-B e NPR-C ed una up-regulation del sottotipo recettoriale NPR-A, sebbene non significative. I livelli di espressione della pre-proET-1 e dei sottotipi recettoriali ET-A ed ET-B non hanno evidenziato differenze significative nei due gruppi di ratti analizzati, mentre è stata osservata una diminuzione significativa negli O per quanto riguarda l’ECE-1 (p=0,02). Nei ratti obesi è stato osservato un aumento significativo dei livelli plasmatici di T3 (p=0,01), T4 (p=0,02) e una contemporanea diminuzione dei livelli plasmatici di TSH (p=0,005). I fattori infiammatori analizzati, IL-6 e TNF-α, non subiscono alterazioni a livello di espressione, nei due gruppi analizzati. E’ stata osservata una correlazione positiva tra CNP e TNF-α (p=0,05), tra BNP e NPR-A (p=0,009), tra BNP e NPR-B (p=0,011) e tra ET-1 e i rispettivi sottotipi recettoriali ((p=0,001). Quando i due gruppi vengono ulteriormente suddivisi in ratti a digiuno e con iperglicemia l’espressione del BNP continua a risultare inferiore negli obesi rispetto ai controlli ma con livelli significativi solo per i ratti iperglicemici (p=0,04); il sottotipo recettoriale NPR-B presenta livelli di espressione identici tra COdig e Odig, mentre è possibile osservare una differenza più marcata tra COiper e Oiper. Anche l’ET-1 subisce variazioni: la prepro-ET-1 diminuisce marcatamente sia negli Odig che negli Oiper, con differenze significative tra COdig e Odig (p=0,04). Un ulteriore differenza significativa è possibile osservarla anche tra COdig e COiper (p=0,03). Si osserva un cambiamento anche nel profilo tiroideo: i livelli plasmatici del TSH negli Oiper. diminuiscono significativamente rispetto al COiper (p<0,0001), mentre negli Odig è possibile osservare un andamento inverso dei livelli di TSH. Inoltre è interessante notare gli elevati livelli di TSH nei COiper rispetto ai COdig (p<0,0001), mentre i livelli della T3 e della T4 risultano aumentati in entrambi i gruppi negli obesi con livelli significativi per la T4 tra COdig e Odig (p=0,03). Conclusioni: I dati ottenuti in questo lavoro di tesi sottolineano che le molteplici variabili che subentrano in un così complesso sistema possono influire contrastandosi o sinergizzandosi vicendevolmente. La comprensione dei meccanismi coinvolti nella modulazione del sistema dei PN e dell’ET-1 nell’obesità è un utile punto di partenza per studi futuri volti a identificare nuove strategie terapeutiche per la cura della sindrome cardiometabolica

Regulation of protein function by S-nitrosation and S-glutathionylation: processes and targets in cardiovascular pathophysiology

International audienceDecades of chemical, biochemical and patho-physiological research have established the relevance of post-translational protein modifications induced by processes related to oxidative stress, with critical reflections on cellular signal transduction pathways. A great deal of the so-called 'redox regulation' of cell function is in fact mediated through reactions promoted by reactive oxygen and nitrogen species on more or less specific ami-noacid residues in proteins, at various levels within the cell machinery. Modifications involving cysteine residues have received most attention, due to the critical roles they play in determining the structure/function correlates in proteins. The peculiar reactivity of these residues results in two major classes of modifications, with incorporation of NO moieties (S-nitrosation, leading to formation of protein S-nitrosothiols) or binding of low molecular weight thiols (S-thionylation, i.e. in particular S-glutath-ionylation, S-cysteinylglycinylation and S-cysteinylation). A wide array of proteins have been thus analyzed in detail as far as their susceptibility to either modification or both, and the resulting functional changes have been described in a number of experimental settings. The present review aims to provide an update of available knowledge in the field, with a special focus on the respective (sometimes competing and antagonistic) roles played by protein S-nitrosations and S-thionylations in biochemical and cellular processes specifically pertaining to pathogenesis of cardiovascular diseases

Effect of the three-dimensional organization of liver cells on the biogenesis of the γ-glutamyltransferase fraction pattern

Context Four gamma-glutamyltransferase (GGT) fractions with different molecular weights (big-, medium-, small- and free-GGT) are detectable in human plasma. Objective Verify if liver cells can release all four GGT fractions and if the spatial cell organization influences their release. Methods Hepatoma (HepG2) and melanoma (Me665/2/60) cells were cultured as monolayers or spheroids. GGT released in culture media was analysed by gel-filtration chromatography. Results HepG2 and Me665/2/60 monolayers released the b-GGT fraction, while significative levels of s-GGT and f-GGT were detectable only in media of HepG2-spheroids. Bile acids alone or in combination with papain promoted the conversion of b-GGT in s-GGT or f-GGT, respectively. Conclusions GGT is usually released as b-GGT, while s-GGT and f-GGT are likely to be produced in the liver extracellular environment by the combined action of bile acids and proteases