Background-RAGE (receptor for advanced glycation end products [AGEs]) plays a role in diabetic atherosclerosis.Recently, we have demonstrated enhanced expression of cyclooxygenase-2 and PGE synthase-1 (COX-2/mPGES-1) in human symptomatic plaques, and provided evidence that it is associated with metalloproteinase (MMP)-induced plaque rupture. However, the specific transmembrane signaling pathway(s) influencing plaque COX-2/mPGES-1 expression is unknown. The aim of this study was to characterize RAGE expression in human plaques and to correlate it with the inflammatory infiltration, COX-2/mPGES-1 and MMP expression, and with clinical evidence of diabetes. Methods and Results-Plaques obtained from 60 patients undergoing carotid endarterectomy were divided into diabetic and nondiabetic according to clinical evidence of type 2 diabetes. Plaques were subjected to analysis of RAGE, NF-B, COX-2/mPGES-1, MMP-2 and MMP-9, lipid and oxidized LDL (oxLDL) content, and collagen content by immunohistochemistry and Western blot, whereas zymography was used to detect MMP activity. Immunohistochemistry was used to identify CD68ϩ macrophages, CD3ϩ T-lymphocytes, smooth muscle cells (SMCs), and HLA-DRϩ inflammatory cells. Diabetic plaques had more (PϽ0.0001) macrophages, T-lymphocytes, and HLA-DRϩ cells, more (PϽ0.0001) immunoreactivity for RAGE, activated NF-B, COX-2/mPGES-1, and MMPs, increased (PϽ0.0001) gelatinolytic activity, reduced (PϽ0.0001) collagen content, and increased (PϽ0.0001) lipid and oxLDL content. Interestingly, RAGE, COX-2/mPGES-1, and MMP expression was linearly correlated with plasma level of HbA1c. Conclusions-In conclusion, this study demonstrates in humans that RAGE overexpression is associated with enhanced inflammatory reaction and COX-2/mPGES-1 expression in diabetic plaque macrophages, and this effect may contribute to plaque destabilization by inducing culprit metalloproteinase expression.

A number of studies suggest that cancer stem cells are essential for tumour growth, and failure to target these cells can result in tumour relapse. As this population of cells has been shown to be resistant to radiation and chemotherapy, it is essential to understand their biology and identify new therapeutic approaches. Targeting cancer metabolism is a potential alternative strategy to counteract tumour growth and recurrence. Here we applied a proteomic and targeted metabolomic analysis in order to point out the main metabolic differences between breast cancer cells grown as spheres and thus enriched in cancer stem cells were compared with the same cells grown in adherent differentiating conditions. This integrated approach allowed us to identify a metabolic phenotype associated with the stem-like condition and shows that breast cancer stem cells (BCSCs) shift from mitochondrial oxidative phosphorylation towards fermentative glycolysis. Functional validation of proteomic and metabolic data provide evidences for increased activities of key enzymes of anaerobic glucose fate such as pyruvate kinase M2 isoform, lactate dehydrogenase and glucose 6-phopshate dehydrogenase in cancer stem cells as well as different redox status. Moreover, we show that treatment with 2-deoxyglucose, a well known inhibitor of glycolysis, inhibits BCSC proliferation when used alone and shows a synergic effect when used in combination with doxorubicin. In conclusion, we suggest that inhibition of glycolysis may be a potentially effective strategy to target BCSCs.

Aging mitochondrial dysfunction evaluated by metabolomic profiling is associated with MACEs, independently of standard predictors.

Summary. Background: Even though the acetylation of platelet cyclooxygenase (COX)-1 at serine-529 is the direct mechanism of action of low-dose aspirin, its antiplatelet effect has been characterized using indirect indexes of COX-1 activity. Objectives: We performed a clinical study with enteric-coated low-dose aspirin (EC-aspirin), in healthy subjects, to evaluate the effects on the extent and duration of platelet COX-1 acetylation, using a novel proteomic strategy for absolute protein quantification (termed AQUA), as compared with traditional pharmacokinetic and pharmacodynamic parameters. Subjects and methods: In a phase I, single-arm, open-label study of EC aspirin (100 mg day À1 ) administered to 24 healthy subjects, we compared, over a 24 h-period on day 1 and 7, % platelet acetylated COX-1 (AceCOX-1) with traditional pharmacokinetic and pharmacodynamics [i.e. serum thromboxane (TX) B 2 , platelet function by monitoring CEPI(collagen/epinephrine) closure time (CT) using whole-blood PFA-100 and urinary excretion of 11-dehydro-TXB 2 ] parameters. Results: Acetylation of platelet COX-1 was measurable before detection of aspirin levels in the systemic circulation and increased in a cumulative fashion upon repeated dosing. After the last dose of EC-aspirin, %AceCOX-1, serum TXB 2 and CEPI-CT values were maximally and persistently modified throughout 24 h; they averaged 76 AE 2%, 99.0 AE 0.4% and 271 AE 5 s, respectively. EC-aspirin caused 75% reduction in urinary 11-dehydro-TXB 2 excretion. After chronic dosing with aspirin, the pharmacokinetics of acetylsalicylic acid was completely dissociated from pharmacodynamics. Conclusions: The demonstrated feasibility of quantifying the extent and duration of platelet COX-1 acetylation will allow characterizing the genetic, pharmacokinetic and pharmacodynamic determinants of the inter-individual variability in the antiplatelet response to low-dose aspirin as well as identifying extraplatelet sites of drug action.

Background-Clinical trials have demonstrated that agents that inhibit the angiotensin II pathway confer benefit beyond the reduction of blood pressure alone. However, the molecular mechanism underlying this effect has yet to be investigated. Recently, we have demonstrated enhanced expression of inducible cyclooxygenase (COX) and prostaglandin (PG)E 2 -dependent synthase (COX-2/mPGES-1) in human symptomatic plaques and provided evidence that it is associated with metalloproteinase (MMP)-induced plaque rupture. Thus, the aim of this study was to characterize the effect of the angiotensin II type 1 (AT 1 ) receptor antagonist irbesartan on the inflammatory infiltration and expression of COX-2/mPGES-1 and MMPs in human carotid plaques. Methods and Results-Seventy patients with symptomatic carotid artery stenosis were randomized to irbesartan (300 mg/d) or chlorthalidone (50 mg/d) for 4 months before endarterectomy. Plaques were subjected to analysis of COX-1, COX-2, mPGES-1, MMP-2, and MMP-9, angiotensin II, AT 1 , AT 2 , and collagen content by immunocytochemistry, Western blot, and reverse-transcriptase polymerase chain reaction, whereas zymography was used to detect MMP activity. Immunohistochemistry was also used to identify CD68ϩ macrophages, CD3ϩ T lymphocytes, smooth muscle cells (SMCs), and HLA-DRϩ inflammatory cells. Plaques from the irbesartan group had fewer (PϽ0.0001) macrophages, T lymphocytes, and HLA-DRϩ cells; less (PϽ0.0001) immunoreactivity for COX-2/mPGES-1 and MMPs; reduced (PϽ0.0001) gelatinolytic activity; and increased (PϽ0.0001) collagen content. It is worth noting that COX-2/mPGES-1 inhibition was observed after incubation in vitro with irbesartan but not with the selective AT 2 blockade PD123,319. Conclusions-This study demonstrates that irbesartan decreases inflammation and inhibits COX-2/mPGES-1 expression in plaque macrophages, and this effect may in turn contribute to plaque stabilization by inhibition of MMP-induced plaque rupture.