Angiotensin-converting-enzyme inhibition counteracts angiotensin II-mediated endothelial cell dysfunction by modulating the p38/SirT1 axis

Objective: Oxidative stress has been linked to endothelial dysfunction and angiotensin II stimulates the reactive oxygen species production contributing to several cardiovascular diseases. We have studied the chain of events induced by angiotensin-converting-enzyme (ACE) activation in vascular umbilical vein endothelial cells (HUVECs) by using an ACE inhibitor such as zofenoprilat. Methods: We used specific assay to measure the superoxide anion production, tetrazolium bromide (MTT) assay for cell viability, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay for cell apoptosis, and western blot for protein analysis in the study. Results: Zofenoprilat counteracts the superoxide anion production and cell apoptosis induced by angiotensin I treatment by blocking the extrinsic caspase cascade, NF-kB and p38 activation. p38 inhibitor SB203580 reverted the angiotensin II oxidant effects while the p38 constitutively activation, by MKK6 transfection, abrogated the zofenoprilat effects. Characterizing the zofenoprilat downstream effector we found that zofenoprilat reverted the SirT-1 downregulation induced by angiotensin II. p38 activation by angiotensin II was strictly correlated with SirT1 protein downregulation; SB203580 significantly prevented SirT1 downregulation induced by angiotensin II while the p38 constitutive activation abolished SIRT1 protein basal levels. p38 directly bound SirT1 sequestering it in the cytoplasm. SirT1 inhibition by sirtinol annulled zofenoprilat action while SirT1 overexpression reverted the cytotoxic effects of angiotensin II. Finally, zofenoprilat negatively controlled angiotensin I receptor protein expression through SirT1. Conclusion: The p38-SirT1 axis is found markedly relevant in modulating the cardiovascular benefit deriving from ACE-inhibitors and might represent a novel target for innovative drugs in cardiovascular prevention. © 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins

Effects on the incidence of cardiovascular events of the addition of pioglitazone versus sulfonylureas in patients with type 2 diabetes inadequately controlled with metformin (TOSCA.IT): a randomised, multicentre trial

Background The best treatment option for patients with type 2 diabetes in whom treatment with metformin alone fails to achieve adequate glycaemic control is debated. We aimed to compare the long-term effects of pioglitazone versus sulfonylureas, given in addition to metformin, on cardiovascular events in patients with type 2 diabetes. Methods TOSCA.IT was a multicentre, randomised, pragmatic clinical trial, in which patients aged 50\ue2\u80\u9375 years with type 2 diabetes inadequately controlled with metformin monotherapy (2\ue2\u80\u933 g per day) were recruited from 57 diabetes clinics in Italy. Patients were randomly assigned (1:1), by permuted blocks randomisation (block size 10), stratified by site and previous cardiovascular events, to add-on pioglitazone (15\ue2\u80\u9345 mg) or a sulfonylurea (5\ue2\u80\u9315 mg glibenclamide, 2\ue2\u80\u936 mg glimepiride, or 30\ue2\u80\u93120 mg gliclazide, in accordance with local practice). The trial was unblinded, but event adjudicators were unaware of treatment assignment. The primary outcome, assessed with a Cox proportional-hazards model, was a composite of first occurrence of all-cause death, non-fatal myocardial infarction, non-fatal stroke, or urgent coronary revascularisation, assessed in the modified intention-to-treat population (all randomly assigned participants with baseline data available and without any protocol violations in relation to inclusion or exclusion criteria). This study is registered with ClinicalTrials.gov, number NCT00700856. Findings Between Sept 18, 2008, and Jan 15, 2014, 3028 patients were randomly assigned and included in the analyses. 1535 were assigned to pioglitazone and 1493 to sulfonylureas (glibenclamide 24 [2%], glimepiride 723 [48%], gliclazide 745 [50%]). At baseline, 335 (11%) participants had a previous cardiovascular event. The study was stopped early on the basis of a futility analysis after a median follow-up of 57\uc2\ub73 months. The primary outcome occurred in 105 patients (1\uc2\ub75 per 100 person-years) who were given pioglitazone and 108 (1\uc2\ub75 per 100 person-years) who were given sulfonylureas (hazard ratio 0\uc2\ub796, 95% CI 0\uc2\ub774\ue2\u80\u931\uc2\ub726, p=0\uc2\ub779). Fewer patients had hypoglycaemias in the pioglitazone group than in the sulfonylureas group (148 [10%] vs 508 [34%], p<0\uc2\ub70001). Moderate weight gain (less than 2 kg, on average) occurred in both groups. Rates of heart failure, bladder cancer, and fractures were not significantly different between treatment groups. Interpretation In this long-term, pragmatic trial, incidence of cardiovascular events was similar with sulfonylureas (mostly glimepiride and gliclazide) and pioglitazone as add-on treatments to metformin. Both of these widely available and affordable treatments are suitable options with respect to efficacy and adverse events, although pioglitazone was associated with fewer hypoglycaemia events. Funding Italian Medicines Agency, Diabete Ricerca, and Italian Diabetes Society