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Ruolo di SIRT1 nella regolazione di e-selectine nella sindrome metabolica
Full text linkBackground. Endothelial dysfunction and inflammation play a pathophysi-ological role in the development of metabolic syndrome (MetS) and its cardiovas-cular complications. Recent studies have shown that activation of sirtuin 1 (SIRT1), is reduced in mononuclear cells (PBMC) of patients with MetS, but the causes of this alteration is still undefined. Sirtuins are a family of seven enzymes (SIRT1-7) that are activated by caloric restriction and provide resistance to stress, reduced apoptosis and regulate the cell cycle. The purpose of this study was there-fore a) to study the relationship between expression of SIRT1 and markers of endo-thelial dysfunction (ICAM, VCAM, E-selectin) and inflammation (TNF- α) in pa-tients with MetS; b) to verify, in endothelial cells (HUVECs), the role of SIRT1 in the regulation of E-selectin release induced by TNF-α.
Patients and Methods. We studied 85 healthy volunteers (age 47 ± 0.9, F 64 and F 21), of which 21 met the criteria for MetS according to ATPIII. Gene ex-pression of SIRT1 was determined in mononuclear cells (PBMC) by quantitative real-time PCR analysis and Western Blot. The plasma concentration of ICAM-1, VCAM-1 and E-selectin were analyzed on the Luminex platform (Bio-Plex sys-tem) using the Millipore kit 3-plex Human Panel-1 cardiovascular disease. In vitro, HUVECs were treated with TNF-α (0.1 μg/ml) to measure E-selectin release (Elisa kit), SIRT1 and nuclear factor-kB (NF-kB) expression and NF-kB activation (Im-munoprecipitation and Western Blot). SIRT1 silencing was determined by siRNA for SIRT1. Chromatin immunoprecipitation was performed to determine the bind-ing of NF-kB to the E-selectin promoter after TNF-α stimulation.
Results. Gene and protein expression of SIRT1 were significantly reduced in PBMCs of patients with MetS than non-MetS. The plasma concentration of E-selectin and TNF-α were significantly increased in subjects with MS than non-MS, while ICAM-1 and VCAM-1 were not different in the two groups of subjects. The plasma levels of E-selectin were positively correlated with blood pressure, waist circumference, triglycerides, blood glucose and insulin. SIRT1 gene expression was negatively correlated with plasma E-selectin. In HUVECs, TNF-α induced a maximal release of E-selectin after 24h, increased the level and the activation (ace-tylation) of p65 subunit of NF-kB. These effects were abolished by the presence of resveratrol, an activator of SIRT1, but not by sirtinol, an inhibitor of SIRT1, or in SIRT1-siRNA silencing cells. Finally, chromatin immunoprecipitation analysis demonstrated the binding of p65 subunit of NF-kB to the E-selectin promoter in-duced by TNF-α which was inhibited by resveratrol.
Conclusion The metabolic abnormalities seen in patients with MetS are as-sociated with increased plasma concentrations of E-selectin and a reduction of ex-pression of SIRT1.In HUVECs, the activation of SIRT1 by resveratrol decreases the release of E-selectin by attenuating the activation of NF-kB and the interaction with E-selectin promoter. Therefore, these results suggest a link between endothe-lial dysfunction and the regulation of SIRT1 which plays an important role in MetS.Background. La disfunzione endoteliale e l’infiammazione svolgono un ruolo fisiopatologico fondamentale nello sviluppo della sindrome metabolica (MetS) e delle sue complicanze cardiovascolari. Recenti studi hanno dimostrato che l’espressione della proteina sirtuina 1 (SIRT1), enzima ad attività deacetilasica NAD+-dipendente, è ridotta nei linfomonociti (PBMC) di soggetti affetti da MetS, ma le cause di questa alterazione non sono ancora definite. Le sirtuine sono una famiglia di 7 enzimi (SIRT1-7) che vengono attivati dalla restrizione calorica e danno resistenza allo stress, riducono l’apoptosi e regolano il ciclo cellulare. Scopo dello studio è stato, pertanto, studiare, a) in vivo, la relazione tra l’espressione ge-nica di SIRT1e i markers di disfunzione endoteliale (ICAM, VCAM, E-selectine) e di infiammazione (TNF- α) in soggetti con SM e, b) in vitro, nelle cellule endote-liali (HUVECs) il ruolo di SIRT1 nella regolazione del rilascio di E-selectine sti-molato da TNF- α.
Metodi. In vivo, sono stati studiati 85 soggetti volontari sani (età 47± 0.9, M 64 e F 21), di cui 21 soddisfacevano i criteri di SM secondo l’ATPIII. Il plasma e linfomonociti sono stati ottenuti da ciascun soggetto per la determinazione della espressione genica di SIRT1 (PCR real time) e delle molecole di adesione e di in-fiammazione. La concentrazione plasmatica e nel terreno di coltura di ICAM-1, VCAM-1, E-selectine e TNF-α sono state analizzate su piattaforma Luminex (Bio-Plex System). In vitro, le cellule endoteliali (HUVECs) sono state incubate con TNF-α (0.1 μg/ml) per determinare il rilascio di E-selectine, l’espressione di SIRT1 e del fattore nucleare-kB (NF-kB), e l’attivazione (acetilazione) di NF-kB. Il silenziamento del gene SIRT1 è stato determinato mediante trasfezione delle cel-lule con siRNA per SIRT1. L’immunoprecipitazione della cromatina è stata esegui-ta per misurare il legame tra il fattore trascrizionale NF-kB e il promotore per il gene di E-selectine dopo la stimolazione con TNF-α.
Risultati. L’espressione genica e proteica di SIRT1 sono risultate essere significativamente ridotte nei PBMC dei soggetti affetti da MetS rispetto a quelli non MetS. La concentrazione plasmatica di E-selectine e di TNF-α sono risultate essere significativamente aumentate nei soggetti con SM rispetto a quelli non MetS, mentre ICAM-1 e VCAM-1 non sono risultate differenti nei due gruppi di soggetti. I valori plasmatici di E-selectine si sono correlati positivamente con pres-sione arteriosa, circonferenza vita, trigliceridi, glicemia e con insulinemia. L’espressione genica di SIRT1 si è correlata negativamente con la concentrazione plasmatica di E-selectine. In vitro, l’incubazione con TNF-α ha aumentato il rila-scio di E-selectine e l’espressione e l’attivazione della subunità p65 di NF-kB. Questi effetti sono stati inibiti dalla presenza di resveratrolo, attivatore di SIRT1, ma non dal sirtinolo, inibitore di SIRT1, o dal silenziamento di SIRT1. L’immunoprecipitazione della cromatina ha dimostrato che la subunità p65 di NF-kB si lega al promotore del gene E-selectine e questa interazione viene ridotta dalla presenza di resveratrolo.
Conclusioni Le alterazioni metaboliche presenti nei soggetti con SM si associano ad un incremento della concentrazione plasmatica di E-selectine e ad una riduzione della espressione genica di SIRT1. Nelle cellule endoteliali l’attivazione di SIRT1 mediata da resveratrolo diminuisce il rilascio di E-selectine mediante la riduzione dell’attivazione di NF-kB e dell’interazione con il promotore del gene E-selectine. Pertanto, questi risultati suggeriscono un legame tra disfunzione endoteliale e la regolazione di SIRT1 nella sindrome metabolica
Positive cardiac inotropic effect of albumin infusion in rodents with cirrhosis and ascites: molecular mechanisms
No full textAbnormal calcium release by angiotensin II and growth rate in skin fibroblasts from type 1 diabetic patients with diabetic nephropathy.
No full textRed blood cell sodium - proton exchange (NHE) and sodium - lithium exchanger (NLE) in hypertension and diabetes mellitus: kinetic effects of insulin and insulin resistance.
No full textEndothelin-1-induced arachidonic acid release by cytosolic phospholipase A(2) activation in rat vascular smooth muscle via extracellular signal-regulated kinases pathway
No full textThe present study investigates whether endothelin-1 (ET-1), like noradrenaline (NA), stimulates the release of arachidonic acid (AA) via cytosolic phospholipase A2 (cPLA2) in rat tail artery. In tail artery segments labelled with [3H]AA, ET-1-induced AA release in a concentration-dependent manner with an EC50 of 1.3 nM. The effect of ET-1 was inhibited by bosentan and was insensitive to BQ788, suggesting the involvement of ETA receptor. The stimulation of AA release induced by ET-1 was prevented by arachydonyl trifluoromethyl ketone (AACOCF3), a selective inhibitor of cPLA2 and not by RHC80267, a diacylglycerol lipase inhibitor. Furthermore, PD98059, inhibitor of mitogen-activated protein kinase kinase (MEK) cascade and calphostin C, a protein kinase C (PKC) inhibitor, prevented the stimulation of AA release induced by ET-1 and NA. Immunoblotting of the cytosolic fraction of rat tail arteries stimulated with ET-1 or NA showed an increase in extracellular signal-regulated kinases (ERKs) phosphorylation and this effect was abolished by calphostin C treatment. These findings show that in rat tail artery ET-1 and NA induce a sequential activation of protein kinase C and extracellular signal-regulated kinases that results in stimulation of AA release via cPLA2 activation. This may represent a general pathway by which G-proteins coupled receptors stimulate AA release and its metabolites in vascular smooth muscle
The Li+/Na+ exchange in hypertension
No full textThe red cell membrane Li+/Na+exchange is a heteroexchange that operates in either direction across the cell membrane. It binds either Li+ or Na+ on one side of the membrane and it exchanges the transported species for either Li+ or Na+ on the opposite side in a stoichiometric ratio of 1:1. In the population, Li+/Na+exchange is unimodally distributed but skewed to the right. Such distribution results from superimposition of two normal distributions. Many laboratories have shown that red-cell Li+/Na+ exchange is increased in patients with essential hypertension, compared with normotensive controls. Among the various alterations of cell membrane cation transport reported in hypertension, the increase of red-cell Li+/Na+ exchange has been most widely investigated and confirmed. Moreover, increased Li+/Na+ exchange has been found in some clinical conditions related to hypertension, such as overweight and diabetes. Among diabetic patients, Li+/Na+ exchange is particularly high in patients with nephropathy, hypertension, and microalbuminuria, leading to the hypothesis that it can be considered a cellular marker of the risk of developing diabetic nephropathy. Furthermore, it is associated with severe and drug-resistant hypertension, insulin resistance, vascular and cardiac hypertrophy, hyperlipidemia, obesity, family history of hypertension, and of major cardiovascular accidents suggesting that high Li+/Na+ exchange could be a biochemical marker for increased cardiovascular risk. Regardless of its the pathophysiological significance, its measurement could be of clinical use as an intermediate phenotype of increased cardiovascular risk
Ruolo della MAP chinasi nell'attivazione della fosfolipasi A2 citosolica indotta da noradrenalina ed endotelina-1 nel muscolo liscio vascolare
No full textG-Protein β3-Subunit Gene C825T Polymorphism and Cardiovascular Risk: An Updated Review
No full textHypertension is a common disorder of multifactorial origin that constitutes a major risk factor for cardiovascular events such as stroke and myocardial infarction. The subunits of the heterotrimeric G proteins are attractive candidate gene products for susceptibility to hypertension, obesity and insulin resistance syndrome. A polymorphism (825C/T) in exon 10 of the GNB3 gene, encoding for the Gβ3 subunit, has been described. The 825T allele is associated with alternative splicing of the gene and formation of a truncated but functionally active β3 subunit. Many studies have investigated whether carriers of the 825T allele are at increased risk for hypertension, obesity, insulin-resistance and left ventricular hypertrophy with apparently conflicting results. The present review demonstrates that GNB3 825T allele is a useful genetic marker for better defining the risk profile of hypertensive patients, as it is associated with increased risk of stroke and myocardial infarction in longitudinal studies in Caucasians
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