Ludovica Perri

Oxidative stress: New insights on the association of non-alcoholic fatty liver disease and atherosclerosis.

Non-alcoholic fatty liver disease (NAFLD) represents the most common and emerging chronic liver disease worldwide. It includes a wide spectrum of liver diseases ranging from simple fatty liver to non-alcoholic steatohepatitis (NASH), which may progress to fibrosis and more severe liver complications such as cirrhosis, hepatocellular carcinoma and liver mortality. NAFLD is strongly associated with obesity, insulin resistance, hypertension, and dyslipidaemia, and is now regarded as the liver manifestation of the metabolic syndrome. The increased mortality of patients with NAFLD is primarily a result of cardiovascular disease and, to a lesser extent, to liver related diseases. Increased oxidative stress has been reported in both patients with NAFLD and patient with cardiovascular risk factors. Thus, oxidative stress represents a shared pathophysiological disorder between the two conditions. Several therapeutic strategies targeting oxidative stress reduction in patients with NAFLD have been proposed, with conflicting results. In particular, vitamin E supplementation has been suggested for the treatment of non-diabetic, non-cirrhotic adults with active NASH, although this recommendation is based only on the results of a single randomized controlled trial. Other antioxidant treatments suggested are resveratrol, silybin, L-carnitine and pentoxiphylline. No trial so far, has evaluated the cardiovascular effects of antioxidant treatment in patients with NAFLD. New, large-scale studies including as end-point also the assessment of the atherosclerosis markers are needed.

Obesity and Hypercholesterolemia are Associated with NOX2 Generated Oxidative Stress and Arterial Dysfunction

OBJECTIVE To analyze the interplay among oxidative stress, NOX2, the catalytic core of nicotinamide-adenine dinucleotide phosphate oxidase, and endothelial dysfunction in children with obesity and/or hypercholesterolemia. STUDY DESIGN We performed a cross-sectional study comparing flow-mediated arterial dilation (FMD), oxidized low-density lipoprotein, and urinary excretion of isoprostanes (8-iso-PGF2α), as markers of oxidative stress, and NOX2 activity, as assessed by blood levels of soluble NOX2-dp (sNOX2-dp), in a population of 100 children, matched for age and sex, including 40 healthy subjects (HS), 20 children with hypercholesterolemia (HC), 20 obese children (OC), and 20 children with coexistence of hypercholesterolemia and obesity (HOC). RESULTS HOC had higher sNOX2-dp and oxidized low-density lipoprotein levels compared with HS, HC, and OC. HC, OC, and HOC had lower FMD values compared with HS. Urinary 8-iso-PGF2α excretion was higher in HOC compared with HS. FMD was inversely correlated with sNOX2-dp levels (r = -0.483; P < .001) and with the number of cardiovascular risk factors (r = -0.617; P < .001). Multiple linear regression analysis showed that the number of cardiovascular risk factors was the only independent predictive variable associated with FMD (β: -0.585; P < .001; R(2) = 35%) and sNOX2-dp (β: 0.587; P < .001; R(2) = 34%). CONCLUSION The study suggests that NOX2-generating oxidative stress may have a pathogenic role in the functional changes of the arterial wall occurring in HOC.

DARK CHOCOLATE INHIBITS PLATELET ISOPROSTANES VIA NOX2 DOWN- REGULATION IN SMOKERS

Summary.  Background: Dark chocolate is reported to decrease platelet activation but the underlying mechanism is still undefined. Dark chocolate is rich in polyphenols that could exert an antiplatelet action via inhibition of oxidative stress. The aim of the present study was to assess if dark chocolate inhibits platelet reactive oxidant species (ROS) formation and platelet activation. Methods: Twenty healthy subjects (HS) and 20 smokers were randomly allocated to receive 40 g of dark (cocoa > 85%) or milk chocolate (cocoa < 35%) in a cross‐over, single‐blind study. There was an interval of 7 days between the two phases of the study. At baseline and 2 h after chocolate ingestion, platelet recruitment (PR), platelet ROS, platelet isoprostane 8‐ISO‐prostaglandin F2α (8‐iso‐PGF2α), Thromboxane (TxA2) and platelet activation of NOX2, the catalytic sub‐unit of NADPH oxidase, and serum epicatechin were measured. Results: Compared with HS, smokers showed enhanced PR, platelet formation of ROS and eicosanoids and NOX2 activation. After dark chocolate, platelet ROS (−48%, P < 0.001), 8‐iso‐PGF2α (−10%, P < 0.001) and NOX2 activation (−22%, P < 0.001) significantly decreased; dark chocolate did not affect platelet variables in HS. No effect of milk chocolate was detected in both groups. Serum epicatechin increased after dark chocolate in HS (from 0.454 ± 0.3 nm to 118.3 ± 53.7 nm) and smokers (from 0.5 ± 0.28 nm to 120.9 ± 54.2 nm). Platelet incubation with 0.1–10 μm catechin significantly reduced PR, platelet 8‐iso‐PGF2α and ROS formation and NOX2 activation only in platelets from smokers. Conclusions: Dark chocolate inhibits platelet function by lowering oxidative stress only in smokers; this effect seems to be dependent on its polyphenolic content.

NOX2-mediated arterial dysfunction in smokers: acute effect of dark chocolate

Background Cocoa seems to exert artery dilatation via oxidative stress inhibition but the mechanism is still unclear. Objectives To investigate whether in smokers, dark chocolate elicits artery dilatation via down-regulation of NOX2, the catalytic core of NADPH oxidase. Methods Flow-mediated dilatation (FMD), oxidative stress (as assessed by urinary isoprostanes excretion), nitric oxide generation (as assessed by serum levels of nitrite/nitrate (NOx)), NOX2 activity (as assessed by blood levels of soluble NOX2 derived peptide (sNOX2-dp)) and serum epicatechin were studied in 20 smokers and 20 healthy subjects (HS) in a crossover, single-blind study. Patients were randomly allocated to 40 g dark chocolate (>85% cocoa) or 40 g of milk chocolate (≤35% cocoa). FMD, urinary isoprostanes, NOx and sNOX2-dp were assessed at baseline and 2 h after chocolate ingestion. Results Smokers had lower FMD and NOx and higher sNOX2-dp compared to HS. After dark chocolate intake, urinary isoprostanes and sNOX2-dp significantly decreased and FMD and NOx significantly increased in smokers but not in HS. No changes of the above variables were observed after milk chocolate intake. Multiple linear regression analysis showed that in smokers the only independent predictive variable associated with a change in FMD was a change in sNOX2-dp. Serum epicatechin increased in either group only after dark chocolate intake, reaching values higher than 0.1 μM. Platelets from smokers (n=5), but not from HS (n=5), showed lower p47phox translocation to platelet membrane and higher NOx when incubated with 0.1–10 μM epicatechin. Conclusion Results suggest that in smokers, cocoa enhances artery dilatation by lowering of NOX2 activation.

Dark Chocolate Acutely Improves Walking Autonomy in Patients With Peripheral Artery Disease

Background NOX‐2, the catalytic subunit of NADPH oxidase, has a key role in the formation of reactive oxidant species and is implicated in impairing flow‐mediated dilation (FMD). Dark chocolate exerts artery dilatation via down‐regulating NOX2‐mediated oxidative stress. The aim of this study was to investigate whether dark chocolate improves walking autonomy in peripheral artery disease (PAD) patients via an oxidative stress‐mediated mechanism. Methods and Results FMD, serum levels of isoprostanes, nitrite/nitrate (NOx) and sNOX2‐dp, a marker of blood NOX2 activity, maximal walking distance (MWD) and maximal walking time (MWT) were studied in 20 PAD patients (14 males and 6 females, mean age: 69±9 years) randomly allocated to 40 g of dark chocolate (>85% cocoa) or 40 g of milk chocolate (≤35% cocoa) in a single blind, cross‐over design. The above variables were assessed at baseline and 2 hours after chocolate ingestion. Dark chocolate intake significantly increased MWD (+11%; P<0.001), MWT (+15%; P<0.001), serum NOx (+57%; P<0.001) and decreased serum isoprostanes (−23%; P=0.01) and sNOX2‐dp (−37%; P<0.001); no changes of the above variables were observed after milk chocolate intake. Serum epicatechin and its methylated metabolite significantly increased only after dark chocolate ingestion. Multiple linear regression analysis showed that Δ of MWD was independently associated with Δ of MWT (P<0.001) and Δ of NOx (P=0.018). In vitro study demonstrated that HUVEC incubated with a mixture of polyphenols significantly increased nitric oxide (P<0.001) and decreased E‐selectin (P<0.001) and VCAM1 (P<0.001). Conclusion In PAD patients dark but not milk chocolate acutely improves walking autonomy with a mechanism possibly related to an oxidative stress‐mediated mechanism involving NOX2 regulation. Clinical Trial Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT01947712.

NOX2 up-regulation is associated with artery dysfunction in patients with peripheral artery disease.

OBJECTIVE Oxidative stress seems to play a role in impairing flow-mediated dilation (FMD) in patients with peripheral artery disease (PAD) but the underlying mechanism is still undefined. We evaluated whether NOX2, the catalytic core of NADPH oxidase, the most important producer of reactive oxidant species (ROS), is implicated in impairing FMD. METHODS We measured FMD, urinary isoprostanes, a marker of oxidative stress, nitric oxide generation by serum levels of nitrite/nitrate (NOx), and serum levels of soluble NOX2-derived peptide (sNOX2-dp), a marker of NOX2 activation, in 50 PAD patients and 50 controls. Also, we performed an interventional cross-over study to assess if propionyl-L-carnitine (PLC) (6g/day), vs. placebo, was able to affect FMD via an oxidative stress-mediated mechanism. RESULTS Compared to controls, patients with PAD had enhanced sNOX2-dp and isoprostanes and reduced NOx and FMD. Multiple linear regression analysis showed that FMD was independently associated with sNOX2-dp. After PLC infusion FMD increased while sNOX2-dp and isoprostanes significantly decreased; no changes were observed after placebo. In vitro study by incubating platelets or white cells with PLC demonstrated a significant inhibition of p47(phox) translocation on cellular surface and ROS generated by NOX2 activation. CONCLUSION This study suggests that in PAD patients ROS generated by NOX2 contribute to reduce FMD and that the administration of an antioxidant is able to improve arterial dilatation via NOX2 inhibition.

Endothelial dysfunction and oxidative stress in children with sleep disordered breathing: Role of NADPH oxidase

OBJECTIVE Oxidative stress plays a crucial role in impairing endothelial function in sleep disordered breathing (SDB) but the underlying mechanism is still undefined. The objective of this study was to evaluate the interplay between oxidative stress, assessed by serum isoprostanes (8-iso-PGF2α) and soluble NOX2-dp (sNOX2-dp), and endothelial function, assessed by flow-mediated dilation (FMD), in children with SDB and healthy controls (HC). METHODS One-hundred forty-four children including 45 with primary snoring (PS), 22 with obstructive sleep apnea (OSA) and 67 HC were recruited in this study; in 15 out of 22 OSA children FMD, serum 8-iso-PGF2α and sNOX2-dp were assessed before and after one month post adeno-tonsillectomy (AT). RESULTS Compared with HC, OSA and PS children had significantly higher sNOX2-dp and serum 8-iso-PGF2α levels and lower FMD; compared with PS, FMD was significantly lower in OSA children. No significant difference for sNOX2-dp and serum 8-iso-PGF2α was observed between OSA and PS children. FMD was inversely correlated with sNOX2-dp levels (p<0.001) and with serum 8-iso-PGF2α (p<0.001). In multiple linear regression analysis, sNOX2-dp (p<0.001) and serum 8-iso-PGF2α (p<0.001) were the only independent predictive variables associated with FMD. AT significantly decreased sNOX2-dp and serum 8-iso-PGF2α levels (from 38.2±8.8 to 22.4±11.1 pg/ml, p<0.001, and from 281.4±69.7 to 226.0±66.4 pg/ml, p<0.001, respectively); conversely, FMD significantly increased after AT in OSA children (from 3.0±1.5 to 8.0±2.8%, p<0.001). CONCLUSION This study suggests that NOX2-derived oxidative stress is involved in artery dysfunction in SDB children. Such hypothesis is reinforced by FMD improvement after AT coincidentally with oxidative stress lowering. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT02247167.

Myocardial infarction and atrial fibrillation: different impact of anti-IIa vs anti-Xa new oral anticoagulants: a meta-analysis of the interventional trials.

PURPOSE The aim of this study was to perform a meta-analysis, in patients with atrial fibrillation, to evaluate the impact of new oral anticoagulants (NOACs) anti-IIa and anti-Xa vs warfarin on myocardial infarction incidence. METHODS A meta-analysis of double blind randomized controlled trials (RCTs) was performed. RESULTS Four RCT studies including 84,439 patients were selected. Compared with warfarin, no significant reduction of myocardial infarction was observed with anti-Xa drugs; conversely, myocardial infarction was significantly increased with anti-IIa NOAC (1.45% vs 1.04% respectively; RR: 1.38; 95% CI, 1.1-1.7; p=0.005). CONCLUSIONS This meta-analysis suggests that in patients with atrial fibrillation, compared to standard therapy with warfarin, treatment with anti-IIa NOAC is associated with a higher incidence of myocardial infarction.

Impact of new oral anticoagulants on gastrointestinal bleeding in atrial fibrillation: A meta-analysis of interventional trials

BACKGROUND New oral anticoagulants represent an alternative to standard therapy with vitamin K antagonists but data regarding gastrointestinal bleeding are still unclear. AIMS To investigate if new oral anticoagulants are associated with an enhanced risk of gastrointestinal bleeding vs warfarin in patients with atrial fibrillation. METHODS Meta-analysis of phase three randomized controlled trials to compare the incidence of gastrointestinal bleeding in atrial fibrillation patients treated with new oral anticoagulants (apixaban, dabigatran, edoxaban and rivaroxaban) vs warfarin. RESULTS Four studies including 71,302 patients were selected. Compared with warfarin, new oral anticoagulants significantly increased gastrointestinal bleeding (RR: 1.23; 95% CI 1.03-1.46; p=0.01). Rivaroxaban (RR: 1.46; 95% CI 1.2-1.8; p<0.001) and high dosages of edoxaban (RR: 1.22; 95% CI 1.01-1.47; p=0.038) and dabigatran (RR: 1.50; 95% CI 1.20-1.88; p<0.001) significantly increased gastrointestinal bleeding while a null effect was detected with apixaban. CONCLUSIONS This meta-analysis suggests that rivaroxaban and high dosages of dabigatran and edoxaban should be avoided in patients at high risk of gastrointestinal bleeding.

Upstream therapy with statin and recurrence of atrial fibrillation after electrical cardioversion. Review of the literature and meta-analysis

BackgroundAtrial fibrillation (AF) is the most common sustained arrhythmia observed in clinical practice. Electrical cardioversion (EC) is commonly used to restore and maintain sinus rhythm but it is characterized by high rate of recurrences. Several trials analyzed the effects of statins to reduce the recurrences in AF with contradictory results.MethodsWe performed a meta-analysis of the interventional trials with statins in patients with persistent AF to evaluate recurrences after EC. Only randomized controlled trials were included in the analysis. Data sources included: Medline, ISI Web of Science, SCOPUS and Cochrane database (up to June 2012). Data extraction was performed by three authors. Study-specific odds ratios (ORs) were combined using fixed-effects model.ResultsSix studies with 515 patients were included in the analysis. Statins used in the selected trials were: atorvastatin (at dosages ranging from 10 to 80 mg/day), pravastatin (40 mg/day) and rosuvastatin (20 mg/day). AF recurrence after EC occurred in 108/258 (41.8%) of patients treated with statins and in 132/257 (51.3%) patients not on treatment with statins. Compared with control, recurrences were significantly reduced with statin treatment (O.R.: 0.662; 95% C.I., 0.45-0.96; p=0.03); statin treatment was associated with an absolute risk reduction of 0.095 and a number needed to treat of 11.ConclusionsThis review suggests that statin therapy was significantly associated with a decreased risk of recurrence in patients with persistent AF after EC.