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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.

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 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.

Intravenous Ascorbic Acid Infusion Improves Myocardial Perfusion Grade During Elective Percutaneous Coronary Intervention. Relationship With Oxidative Stress Markers

OBJECTIVES Our goal was to explore whether antioxidant vitamin C infusion is able to affect the microcirculation perfusion in patients undergoing elective percutaneous coronary intervention for stable angina. BACKGROUND Periprocedural myocardial injury in the setting of elective percutaneous coronary intervention is associated with increased risk of death, recurrent infarction, and revascularization at follow-up. Despite excellent epicardial blood flow, impaired microcirculatory reperfusion may persist and increases the risk of vascular recurrences. Post-percutaneous coronary intervention induced-oxidative stress is one of the potential mechanisms accounting for impaired perfusion. METHODS Fifty-six patients were enrolled in a prospective, single-center, randomized study comparing 1 g vitamin C infusion (16.6 mg/min, over 1 h before percutaneous coronary intervention) versus placebo. RESULTS At the baseline, Thrombolysis In Myocardial Infarction (TIMI) myocardial perfusion grade <2 was observed in 89% and in 86% of patients randomized to the placebo or vitamin C infusion group, respectively (p > 0.05). After percutaneous coronary intervention, these percentages decreased in the placebo group (32%) and in greater measure in the vitamin C group (4%, p < 0.01). Complete microcirculatory reperfusion (TIMI myocardial perfusion grade = 3) was achieved in 79% of the vitamin C-treated group compared with 39% of the placebo group (p < 0.01); 8-hydroxy-2-deoxyguanosine (p < 0.002) and 8-iso-prostaglandin F(2alpha) (p < 0.02) plasma levels significantly increased in the placebo group while they were significantly reduced in the vitamin C-treated group (p < 0.0001). TIMI myocardial perfusion grade changes from the baseline showed significant correlation with 8-hydroxy-2-deoxyguanosine (p < 0.006) or 8-iso-prostaglandin F(2alpha) (p < 0.01) plasma levels changes. CONCLUSIONS In patients undergoing elective percutaneous coronary intervention, impaired microcirculatory reperfusion is improved by vitamin C infusion suggesting that oxidative stress is implicated in such a phenomenon.

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.

Reduced Lysosomal Acid Lipase Activity in Adult Patients With Non-alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is characterized by intra-hepatic fat accumulation and mechanisms involved in its pathogenesis are not fully explained. Lysosomal Acid Lipase (LAL) is a key enzyme in lipid metabolism. We investigated its activity in patients with fatty liver. LAL activity (nmol/spot/h) was measured in 100 adult healthy subjects (HS) and in 240 NAFLD patients. A sub-analysis on 35 patients with biopsy-proven non-alcoholic steatohepatitis (NASH) was performed. Median LAL activity was 1.15 (0.95–1.72) in HS. It was significantly reduced in NAFLD [0.78 (0.61–1.01), p < 0.001 vs. HS]. A further reduction was observed in the subgroup of NASH [0.67 (0.51–0.77), p < 0.001 vs. HS]. Patients with LAL activity below median had higher values of serum total cholesterol (p < 0.05) and LDL-c (p < 0.05), and increased serum liver enzymes (ALT, p < 0.001; AST, p < 0.01; GGT, p < 0.01). At multivariable logistic regression analysis, factors associated with LAL activity below median were ALT (OR: 1.018, 95% CI 1.004–1.032, p = 0.011) and metabolic syndrome (OR: 2.551, 95% CI 1.241–5.245, p = 0.011), whilst statin use predicted a better LAL function (OR: 0.464, 95% CI 0.248–0.866, p = 0.016). Our findings suggest a strong association between impaired LAL activity and NAFLD. A better knowledge of the role of LAL may provide new insights in NAFLD pathogenesis.

Anoxia-reoxygenation enhances platelet thromboxane A2 production via reactive oxygen species-generated NOX2: effect in patients undergoing elective percutaneous coronary intervention.

Objective—Platelets undergoing anoxia-reoxygenation (AR) simultaneously increase reactive oxygen species (ROS) and thromboxane (Tx) B2. Our aim was to assess whether there is an interplay between activation of NOX2, the catalytic subunit of NADPH oxidase, and platelet TxB2 in vitro and in vivo. Methods and Results—Platelets that underwent AR had enhanced ROS. This was associated with NOX2 activation and was inhibited by incubation with NOX2-blocking peptide. AR was associated with TxB2 and isoprostane production, which were inhibited by NOX2-blocking peptide, vitamin C, and the inhibitor of phospholipase A2. Platelet incubation with 100 &mgr;mol/L aspirin fully prevented AR-induced TxA2 but did not affect isoprostane production. We included 56 aspirin-treated patients undergoing elective percutaneous coronary intervention (PCI) who were randomly allocated to receive either placebo or intravenous infusion of 1 g of vitamin C. Blood TxB2, isoprostanes, and soluble NOX2-derived peptide, a marker of systemic NADPH oxidase activation, significantly increased at 60 and 120 minutes after PCI in placebo-treated but not in vitamin C-treated patients. Conclusion—AR is associated with overproduction of platelet TxB2 and isoprostanes, which is dependent on NOX2-dependent ROS generation. Low doses of aspirin are unable to prevent TxB2 formation in patients who undergo PCI.

The polyphenols quercetin and catechin synergize in inhibiting platelet CD40L expression

Thromb Haemost 2005; 94: 888–9 Dear Sir, Moderate consumption of red wine is associated with lower incidence of cardiovascular disease, however, the source of this cardioprotective effect is still uncertain. Acute coronary syndromes are caused by atherosclerotic plaque rupture with ensuing platelet adhesion, aggregation and thrombus formation. Experimental studies in animals demonstrated that red wine inhibits platelet-dependent flow variation and mural thrombosis but the mechanism has not been fully clarified (1, 2). Even if ethanol inhibits platelet function, this effect is not achievable with moderate alcohol consumption (3); therefore, other mechanisms are likely to be implicated in the antithrombotic effect of moderate consumption of wine. Much attention has been recently focused on the possibility that the cardioprotective effect of wine is dependent on polyphenols, its non-alcoholic component. This suggestion is supported by the existence of an inverse association between polyphenol intake and cardiovascular events (4). Polyphenols possess antioxidant property that could inhibit atherosclerotic lesion via inhibition of LDL oxidation and in turn cholesterol accumulation within the macrophages of atherosclerotic plaque (5). However, the antioxidant property of polyphenols could also modulate other aspects of the inflammatory process that are implicated in the pathogenesis of human atherosclerosis.CD40 ligand (CD40L), a member of the tumor necrosis factor family, is a transmembrane protein with pro-inflammatory and pro-thrombotic properties upon interaction with its receptor CD40 (6). Engagement of CD40L with its receptor stimulates the synthesis of adhesion molecules, chemokines and tissue factor, and activates metalloproteinases (6). The role of CD40L in atherogenesis is confirmed by the fact that, in hyperlipidemic mice, anti-CD40L antibodies reduced the atherosclerotic lesion (7). We have recently demonstrated that platelet production of 02 has a key role in the expression of CD40L. Thus, in patients with hereditary deficiency of gp91phox, the central core of NADPH oxidase, platelet CD40L expression was prevented (7). On the basis of this finding we tested the hypothesis that polyphenols could prevent platelet CD40L expression via inhibition of platelet 02. Blood samples were taken from healthy volunteers. No