Simona Bartimoccia

Inherited Human gp91phox Deficiency Is Associated With Impaired Isoprostane Formation and Platelet Dysfunction

Object—Platelet isoprostane 8-ISO-prostaglandin F2&agr; (8-iso-PGF2&agr;), 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-PGF2&agr; in platelets from 8 male patients with hereditary deficiency of gp91phox, the catalytic subunit of NADPH oxidase, and 8 male controls. On stimulation, platelets from controls produced 8-iso-PGF2&agr;, which was inhibited −8% by aspirin and −58% by a specific inhibitor of gp91phox. Platelets from patients with gp91phox hereditary deficiency had normal thromboxane A2 formation but marked 8-iso-PGF2&agr; reduction compared with controls. In normal platelets incubated with a gp91phox inhibitor 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 gp91phox-deficient patients, agonist-induced platelet aggregation was within the normal range, whereas platelet recruitment was reduced compared with controls. Incubation of platelets from gp91phox-deficient patients with 8-iso-PGF2&agr; 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 with l-NAME, an inhibitor of NO synthase. Conclusion—This study provides the first evidence that platelet 8-iso-PGF2&agr; maximally derives from gp91phox activation and contributes to platelet recruitment via activation of gpIIb/IIIa.

Immediate Antioxidant and Antiplatelet Effect of Atorvastatin via Inhibition of Nox2

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 A2, platelet Nox2, Rac1, p47phox, protein kinase C, vasodilator-stimulated phosphoprotein, nitric oxide, and phospholipase A2, 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, p47phox, and protein kinase C, starting 2 hours after administration. Platelet phospholipase A2 and thromboxane A2 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 A2 activation, along with inhibition of platelet recruitment, platelet isoprostanes, and thromboxane A2, 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 A2. 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.

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.

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.

LDL oxidation by platelets propagates platelet activation via an oxidative stress-mediated mechanism

OBJECTIVES Platelets generate oxidized LDL (ox-LDL) via NOX2-derived oxidative stress. We investigated if once generated by activated platelets ox-LDL can propagate platelet activation. METHODS Experiments were performed in platelets from healthy subjects (HS), hyper-cholesterolemic patients and patients with NOX2 hereditary deficiency. RESULTS Agonist-stimulated platelets from HS added with LDL were associated with a dose-dependent increase of reactive oxidant species and ox-LDL. Agonist-stimulated platelets from HS added with a fixed dose of LDL (57.14 μmol/L) or added with homogenized human atherosclerotic plaque showed enhanced ox-LDL formation (approximately +50% and +30% respectively), which was lowered by a NOX2 inhibitor (approximately -35% and -25% respectively). Compared to HS, ox-LDL production was more pronounced in agonist-stimulated platelet rich plasma (PRP) from hyper-cholesterolemic patients but was almost absent in PRP from NOX2-deficient patients. Platelet aggregation and 8-iso-PGF2α-ΙΙΙ formation increased in LDL-treated washed platelets (+42% and +53% respectively) and PRP (+31% and +53% respectively). Also, LDL enhanced platelet-dependent thrombosis at arterial shear rate (+33%) but did not affect platelet activation in NOX2-deficient patients. Platelet activation by LDL was significantly inhibited by CD36 or LOX1 blocking peptides, two ox-LDL receptor antagonists, or by a NOX2 inhibitor. LDL-added platelets showed increased p38MAPK (+59%) and PKC (+51%) phosphorylation, p47(phox) translocation to platelet membrane (+34%) and NOX2 activation (+30%), which were inhibited by ox-LDL receptor antagonists. CONCLUSION Platelets oxidize LDL, which in turn amplify platelet activation via specific ox-LDL receptors; both effects are mediated by NOX2 activation.

NOX2-generated oxidative stress is associated with severity of ultrasound liver steatosis in patients with non-alcoholic fatty liver disease

BackgroundChronic 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.MethodsThe 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.ResultsPatients 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.ConclusionsWe 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.

Rosuvastatin reduces platelet recruitment by inhibiting NADPH oxidase activation

Rosuvastatin increased vascular endothelial NO and attenuated platelet activation after ischemia-reperfusion in mice; nevertheless, the influence of rosuvastatin on the activation of human platelets and the underlying mechanism has never been investigated. In an in vitro study platelets from 8 healthy donors were incubated with scalar concentrations of rosuvastatin (1-10 μM) before activation. Platelet recruitment (PR), that mimics the propagation of platelet aggregation and is dependent upon isoprostane formation, was investigated. PR was inhibited by rosuvastatin in concentration-dependent manner concomitantly with down-regulation of platelet release of the pro-thrombotic molecule CD40L. This effect was associated with lower production of platelet reactive oxygen species (ROS), isoprostane and activation of the glycoprotein IIb/IIIa and was counteracted by exogenous addition of isoprostanes. Conversely, rosuvastatin concentration-dependently increased platelet NO. Platelet isoprostane formation mainly depends from NADPH oxidase. Rosuvastatin concentration-dependently inhibited platelet sNOX2-dp release, a specific marker of NADPH oxidase activation, PKC phosphorylation and p47(phox) translocation from cytosol to membranes. In an ex vivo study 10 hypercolesterolemic patients were randomly allocated to diet or rosuvastatin (20 mg). We observed that as early as 2h after rosuvastatin PR, platelet isoprostanes formation, platelet CD40L and sNOX2-dp decreased while platelet NO increased; no changes were detected in diet-assigned patients. This study shows that in vitro rosuvastatin impairs platelet activation via inhibition of NOX2-derived oxidative stress. This effect, which is associated ex vivo with acute inhibition of platelet activation, suggests that rosuvastatin behaves as an antiplatelet drug.

Low‐grade endotoxemia and platelet activation in cirrhosis

Patients with cirrhosis may display impaired or enhanced platelet activation, but the reasons for these equivocal findings are unclear. We investigated if bacterial lipopolysaccharide (LPS) is implicated in platelet activation. In a cross‐sectional study, conducted in an ambulatory care clinic and hospital, comparing 69 cirrhosis patients and 30 controls matched for sex, age, and atherosclerotic risk factors, serum levels of LPS, soluble cluster of differentiation 40 ligand and p‐selectin (two markers of platelet activation), and zonulin (a marker of gut permeability) were investigated. Ex vivo and in vitro studies were also performed to explore the effect of LPS on platelet activation. Compared to controls, cirrhosis patients displayed higher serum levels of LPS (6.0 [4.0‐17.5] versus 57.4 [43.4‐87.2] pg/mL, P < 0.0001), soluble cluster of differentiation 40 ligand (7.0 ± 2.2 versus 24.4 ± 13.3 ng/mL, P < 0.0001), soluble p‐selectin (14.2 ± 4.05 versus 33.2 ± 15.2 ng/mL, P < 0.0001), and zonulin (1.87 ± 0.84 versus 2.54 ± 0.94 ng/mL, P < 0.006). LPS significantly correlated with zonulin (r = 0.45, P < 0.001). Ex vivo studies showed that platelets from cirrhosis patients were more responsive to the agonists independently from platelet count; this phenomenon was blunted by incubation with an inhibitor of Toll‐like receptor 4. In vitro study by normal platelets showed that LPS alone (50‐150 pg/mL) did not stimulate platelets but amplified platelet response to the agonists; Toll‐like receptor 4 inhibitor blunted this effect. Conclusion: LPS may be responsible for platelet activation and potentially contributes to thrombotic complications occurring in cirrhosis. (Hepatology 2017;65:571‐581).

Serum NOX2 and urinary isoprostanes predict vascular events in patients with atrial fibrillation

There are limited prospective data evaluating the role of urinary F2-IsoP and NOX2 as predictive markers in atrial fibrillation (AF). The aim of this study was to analyse the role of urinary prostaglandin PGF2alpha (8-iso-PGF2α) and NOX2, markers of systemic oxidative stress, in predicting cardiovascular (CV) events and mortality in anticoagulated non-valvular AF patients. This was a prospective study including 1,002 anticoagulated AF patients, followed for a median time of 25.7 months (interquartile range: 14.8-50.9). All major CV events, CV deaths and all-cause deaths were considered as primary outcomes of the study. CV events included fatal/nonfatal ischaemic stroke, fatal/nonfatal myocardial infarction (MI), cardiac revascularisation and transient ischaemic attack (TIA). Oxidative stress biomarkers, such as urinary 8-iso-PGF2α and serum sNOX2-dp, a marker of NOX2 activation, were measured. A CV event occurred in 125 patients (12.5 %); 78 CV deaths and 31 non-CV deaths were registered. 8-iso-PGF2α and sNOX2-dp were correlated (Rs=0.765 p< 0.001). A significant increased cumulative incidence of CV events and CV deaths was observed across tertiles for 8-iso-PGF2α and sNOX2-dp. An increased rate of all-cause death was observed across tertiles of urinary 8-iso-PGF2α. In Cox or Fine and Gray models, 8-iso-PGF2α predicted CV events and CV and non-CV deaths. The addition of tertiles of 8-iso-PGF2α to CHA2DS2-VASc score improved ROC curves for each outcome and NRI for CV events (0.24 [0.06-0.53] p=0.0067). The study shows that in AF patients 8-iso-PGF2α and NOX2 levels are predictive of CV events and total mortality. F2-IsoP may complement conventional risk factors in prediction of CV events.

Extra virgin olive oil use is associated with improved post-prandial blood glucose and LDL cholesterol in healthy subjects

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.