O-linked β-N-acetylglucosamine during hyperglycemia exerts both anti-inflammatory and pro-oxidative properties in the endothelial system

Elevated cellular levels of protein O-linked β-N-acetylglucosamine (O-GlcNAc) through hexosamine biosynthesis pathway (HBP) are suggested to contribute to cardiovascular adverse effects under chronic hyperglycemic condition associated with oxidative stress and inflammation. Conversely, enhancing O-GlcNAc levels have also been demonstrated being protective against myocardial ischemia/reperfusion injury. We recently demonstrated that hyperglycemia increases oxidative stress and HBP flux in endothelial cells and enhances endothelial expression of vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in response to tumor necrosis factor-α (TNFα) through oxidative stress rather than HBP pathway. Here we present further complementary data showing that enhancing O-GlcNAc levels by glucosamine does not mimic hyperglycemia's effect on TNFα-induced endothelial VCAM-1 and ICAM-1 expression. Glucosamine however inhibits ICAM-1 (not VCAM-1) expression and induces superoxide generation in the cells. The results further suggest that increased O-GlcNAc levels do not mediate the enhancing effect of hyperglycemia on the endothelial inflammatory responses to TNFα. In contrast, it exerts certain anti-inflammatory effects accompanied by pro-oxidative properties. Further work should delineate the exact role of HPB pathway in different aspects of cardiovascular functions, especially those of diabetic cardiovascular complications

Hyperactive S6K1 Mediates Oxidative Stress and Endothelial Dysfunction in Aging: Inhibition by Resveratrol

Mammalian target of rapamycin (mTOR)/S6K1 signalling emerges as a critical regulator of aging. Yet, a role of mTOR/S6K1 in aging-associated vascular endothelial dysfunction remains unknown. In this study, we investigated the role of S6K1 in aging-associated endothelial dysfunction and effects of the polyphenol resveratrol on S6K1 in aging endothelial cells. We show here that senescent endothelial cells displayed higher S6K1 activity, increased superoxide production and decreased bioactive nitric oxide (NO) levels than young endothelial cells, which is contributed by eNOS uncoupling. Silencing S6K1 in senescent cells reduced superoxide generation and enhanced NO production. Conversely, over-expression of a constitutively active S6K1 mutant in young endothelial cells mimicked endothelial dysfunction of the senescent cells through eNOS uncoupling and induced premature cellular senescence. Like the mTOR/S6K1 inhibitor rapamycin, resveratrol inhibited S6K1 signalling, resulting in decreased superoxide generation and enhanced NO levels in the senescent cells. Consistent with the data from cultured cells, an enhanced S6K1 activity, increased superoxide generation, and decreased bioactive NO levels associated with eNOS uncoupling were also detected in aortas of old WKY rats (aged 20–24 months) as compared to the young animals (1–3 months). Treatment of aortas of old rats with resveratrol or rapamycin inhibited S6K1 activity, oxidative stress, and improved endothelial NO production. Our data demonstrate a causal role of the hyperactive S6K1 in eNOS uncoupling leading to endothelial dysfunction and vascular aging. Resveratrol improves endothelial function in aging, at least in part, through inhibition of S6K1. Targeting S6K1 may thus represent a novel therapeutic approach for aging-associated vascular disease

Quisto Ovárico Gigante – um Achado Raro

Serous cystadenomas are the main tumours of the ovarian epithelium in postmenopausal women. They can have a slow indolent growth, causing unspecific signs and symptoms, that are frequently not valued by the patients and by health professionals. This leads to a delay in the imaging exams and in the diagnosis. We present a clinical case of an giant abdominal cyst in a postmenopausal woman.Os cistadenomas serosos são o principal tipo tumoral do epitélio ovárico nas mulheres pós-menopáusicas. O seu crescimento pode ser indolente, originando um conjunto de sintomas e sinais inespecíficos, muitas vezes desvalorizados pela doente e pelos profissionais de saúde. Esta desvalorização leva a um atraso nos exames de imagem e no diagnóstico. Apresentamos um caso de um quisto abdominal gigante numa mulher pós-menopausica

Period2 gene mutant mice show compromised insulin-mediated endothelial nitric oxide release and altered glucose homeostasis

Period2 (Per2) is an important component of the circadian clock. Mutation of this gene is associated with vascular endothelial dysfunction and altered glucose metabolism. The aim of this study is to further characterize whole body glucose homeostasis and endothelial nitric oxide (NO) production in response to insulin in the mPer2(Brdm1) mice. We show that mPer2(Brdm1) mice exhibit compromised insulin receptor activation and Akt signaling in various tissues including liver, fat, heart, and aortas with a tissue-specific heterogeneous diurnal pattern, and decreased insulin-stimulated NO release in the aortas in both active and inactive phases of the animals. As compared to wild type (WT) mice, the mPer2(Brdm1) mice reveal hyperinsulinemia, hypoglycemia with lower fasting hepatic glycogen content and glycogen synthase level, no difference in glucose tolerance and insulin tolerance. The mPer2(Brdm1) mice do not show increased predisposition to obesity either on normal chow or high fat diet compared to WT controls. Thus, mice with Per2 gene mutation show altered glucose homeostasis and compromised insulin-stimulated NO release, independently of obesity

Over-expression of an active S6K1 mutant enhances superoxide, decreases NO production and induces premature senescence in young endothelial cells.

<p>Young HUVECs were transduced either with an empty rAd vector as control or with rAd/CMV-HA-S6K1-ca (a constitutively active S6K1 mutant). (<b>A</b>) Immunoblotting analysis of expression of HA-S6K1-ca with anti-HA antibody or antibodies against S6-S235/S236 (p-S6), total S6, and tubulin on day 2 of post transduction. (<b>B</b>) Four days post transduction, cells were subjected to SA-ß-gal staining. (<b>C</b>) MitoSox, DHE, and DAF-2DA staining were performed on day 2 of post transduction. Quantification of the signals is shown in the corresponding lower panels. n = 6. **p<0.01 and ***p<0.001 vs. control cells. Scale bar = 0.2 mm.</p

Over-expression of an active S6K1 mutant causes eNOS uncoupling in endothelial cells.

<p>Young HUVEC cells were transduced either with an empty rAd vector as control or with rAd/CMV-HA-S6K1ca (a constitutively active S6K1 mutant). Two days post transduction, cells were subjected to (<b>A</b>) Immunoblotting analysis of expression of eNOS-dimers, -monomers with anti-eNOS antibody, HA-S6K1-ca with anti-HA antibody. Ponseau S staining served as loading control. Quantification of the eNOS-dimer/monomer ratio is shown in the lower panel. n = 8 in each group. (<b>B</b>) DHE staining was performed 1 hour after L-NAME (1 mmol/L) treatment. Quantification of the signals is shown in the corresponding lower panels. n = 8. **p<0.01 and ***p<0.001 between indicated groups. Scale bar = 0.2 mm.</p

Silencing S6K1 reduces superoxide and enhances NO production in senescent endothelial cells.

<p>Senescent HUVECs were transduced either with rAd/U6-LacZ^shRNA as control or with rAd/U6-S6K1^shRNA. Four days post transduction, cells were subjected to (<b>A</b>) immunoblotting analysis to reveal the effective silencing of S6K1 and (<b>B</b>) MitoSox, DHE, and DAF-2DA staining. Quantification of the signals from four independent experiments of each group is shown in the corresponding lower panels. ***p<0.001 vs. rAd/U6-LacZ^shRNA control cells. Scale bar = 0.2 mm.</p

Increased S6K1 activity in aortas of old rats is inhibited by resveratrol.

<p>(<b>A</b>) SA-ß-gal staining of the endothelium in an aorta segment of a young and an old WKY rat. (<b>B</b>) Immunoblotting analysis of S6-S235/S236 (p-S6) and total S6 in the aortas of young and old rats (n = 9 of each group). (<b>C</b>) The aortas of old rats were treated <i>ex vivo</i> with solvent as control (C), resveratrol (Resv, 10 µmol/L) or rapamycin (Rapa, 20 ng/ml) in Krebs-Ringer bicarbonate solution (37°C, 95% O₂/5% CO₂) for one hour. The lysates were prepared for Immunoblotting analysis of S6-S235/S236 (p-S6) and total S6 levels (n = 3). *p<0.05 and **p<0.01 between indicated groups.</p