Background—: Statins exert an antithrombotic effect in patients at risk of or with acute thrombosis, but no study has investigated whether this effect is immediate and whether there is an underline mechanism. Methods and Results—: Patients with hypercholesterolemia were randomly allocated to a Mediterranean diet with low cholesterol intake (<300 mg/d; n=15) or atorvastatin (40 mg/d; n=15). Oxidative stress, as assessed by serum Nox2 and urinary isoprostanes, and platelet activation, as assessed by platelet recruitment, platelet isoprostanes, and thromboxane A ₂ , platelet Nox2, Rac1, p47 ^phox , protein kinase C, vasodilator-stimulated phosphoprotein, nitric oxide, and phospholipase A ₂ , were determined at baseline and after 2, 24, and 72 hours and 7 days of follow-up. An in vitro study was also performed to see whether atorvastatin affects platelet oxidative stress and activation. The atorvastatin-assigned group showed a significant and progressive reduction of urinary isoprostanes and serum Nox2, along with inhibition of platelet recruitment, platelet isoprostanes, Nox2, Rac1, p47 ^phox , and protein kinase C, starting 2 hours after administration. Platelet phospholipase A ₂ and thromboxane A ₂ significantly decreased and vasodilator-stimulated phosphoprotein and nitric oxide increased after 24 hours. Low-density lipoprotein cholesterol decreased significantly after 72 hours and further declined after 7 days. No changes were observed in the Mediterranean diet group. In vitro experiments demonstrated that atorvastatin dose-dependently inhibited platelet Nox2 and phospholipase A ₂ activation, along with inhibition of platelet recruitment, platelet isoprostanes, and thromboxane A ₂ , and increased vasodilator-stimulated phosphoprotein and nitric oxide. Conclusions—: The study provides the first evidence that atorvastatin acutely and simultaneously decreases oxidative stress and platelet activation by directly inhibiting platelet Nox2 and ultimately platelet isoprostanes and thromboxane A ₂ . These findings provide a rationale for the use of statins to prevent or modulate coronary thrombosis. Clinical Trial Registration—: URL: http://www.clinicaltrials.gov . Unique identifier: NCT01322711.

Object—: Platelet isoprostane 8-ISO-prostaglandin F2α (8-iso-PGF2α), a proaggregating molecule, is believed to derive from nonenzymatic oxidation of arachidonic acid. We hypothesized that NADPH is implicated in isoprostane formation and platelet activation.Methods and Results—: We studied 8-iso-PGF2α in platelets from 8 male patients with hereditary deficiency of gp91^phox, the catalytic subunit of NADPH oxidase, and 8 male controls. On stimulation, platelets from controls produced 8-iso-PGF2α, which was inhibited −8% by aspirin and −58% by a specific inhibitor of gp91^phox. Platelets from patients with gp91^phoxhereditary deficiency had normal thromboxane A₂formation but marked 8-iso-PGF2α reduction compared with controls. In normal platelets incubated with a gp91^phoxinhibitor or with SQ29548, a thromboxane A₂/isoprostane receptor inhibitor, platelet recruitment, an in vitro model of thrombus growth, was reduced by 44% and 64%, respectively; a lower effect (−17%) was seen with aspirin. Moreover, thrombus formation under shear stress (blood perfusion at the wall shear rate of 1500 s⁻¹) was reduced in samples in which isoprostane formation was inhibited by NADPH oxidase inhibitors. In gp91^phox-deficient patients, agonist-induced platelet aggregation was within the normal range, whereas platelet recruitment was reduced compared with controls. Incubation of platelets from gp91^phox-deficient patients with 8-iso-PGF2α dose-dependently (1 to 100 pmol/L) increased platelet recruitment by mobilizing platelet Ca²⁺and activating gpIIb/IIIa; a further increase in platelet recruitment was detected by platelet coincubation withl-NAME, an inhibitor of NO synthase.Conclusion—: This study provides the first evidence that platelet 8-iso-PGF2α maximally derives from gp91^phoxactivation and contributes to platelet recruitment via activation of gpIIb/IIIa.

Objectives: Extra virgin olive oil (EVOO) is a key component of the Mediterranean diet and seems to account for the protective effect against cardiovascular disease. However, the underlying mechanism is still elusive. Design: We tested the effect of EVOO, added to Mediterranean-type meal, on post-prandial glycemic and lipid profile. Subjects: Post-prandial glycemic and lipid profile were investigated in 25 healthy subjects who were randomly allocated in a cross-over design to a Mediterranean-type meal added with or without 10 g EVOO (first study), or Mediterranean-type meal with EVOO (10 g) or corn oil (10 g; second study). Glycemic profile, which included glucose, insulin, dipeptidyl-peptidase-4 (DPP-4) protein and activity, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), and lipid profile, which included, low-density lipoprotein (LDL) cholesterol (LDL-C), oxidized LDL (ox-LDL), triglycerides and high-density lipoprotein (HDL) cholesterol (HDL-C), were analyzed before and 2 h after the meal. Results: In the first study, 2 h after meal, subjects who assumed a meal with EVOO had significantly lower blood glucose (P<0.001), DPP-4 protein (P<0.001) and activity (P<0.001), LDL-C (P<0.001) and ox-LDL (P<0.001) and higher insulin (P<0.05), GLP-1 (P<0.001) and GIP (P<0.05) compared with those without EVOO. The second study showed that compared with corn oil, EVOO improved both glycemic and lipid profile. Thus, a significantly smaller increase of glucose (P<0.05), DPP4 protein (P<0.001) and activity (P<0.05) and higher increase of insulin (P<0.001) and GLP-1 (P<0.001) were observed. Furthermore, compared with corn oil, EVOO showed a significantly less increase of LDL-C (P<0.05) and ox-LDL (P<0.001). Conclusions: We report for the first time that EVOO improves post-prandial glucose and LDL-C, an effect that may account for the antiatherosclerotic effect of the Mediterranean diet.

Background: Chronic oxidative stress is one of the key mechanisms responsible for disease progression in non-alcoholic fatty liver disease. However, so far, few studies reported increased circulating levels of oxidative stress markers in patients with non-alcoholic fatty liver and no study has been performed with newer markers of systemic oxidative stress. The aim was to assess the relationship between urinary 8-iso-prostaglandin F2α and serum soluble NOX2-derived peptide and the severity of liver steatosis in subjects with non-alcoholic fatty liver.Methods: The study was performed in 264 consecutive patients referred for suspected metabolic disease. Steatosis was defined according to Hamaguchi ultrasonographic criteria. Oxidative stress was assessed by urinary 8-iso- prostaglandin F2α and serum soluble NOX2-derived peptide levels.Results: Patients with non-alcoholic fatty liver had higher (p < 0.001) mean values of urinary 8-iso-PGF2α and of serum soluble NOX2-derived peptide, alanine aminotransferase, Cytokeratin-18 and homeostasis model of insulin resistance and lower values of serum adiponectin as compared to those without. Prevalence of metabolic syndrome and of its clinical features was significantly higher in patients with non-alcoholic fatty liver. Same findings were also observed after the exclusion of obese subjects, or subjects with diabetes or with metabolic syndrome and in those not taking statin medication. In addition, the levels of urinary 8-iso-PGF2α were independent predictors of non-alcoholic fatty liver and a strong association of urinary 8-iso-PGF2α and of serum soluble NOX2-derived peptide with the severity of steatosis at ultrasound was also observed.Conclusions: We demonstrated increased markers of oxidative stress in subjects with non-alcoholic fatty liver. Urinary 8-iso-PGF2α and serum soluble NOX2-derived peptide levels were independent from obesity, diabetes and metabolic syndrome and increased with the severity of liver steatosis at ultrasound.

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 NOX 2‐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 sNOX 2‐dp, a marker of blood NOX 2 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 sNOX 2‐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 VCAM 1 ( 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 NOX 2 regulation. Clinical Trial Registration: URL : http://www.clinicaltrials.gov . Unique identifier: NCT 01947712.