Valerio Sanguigni

Short-term treatment with atorvastatin reduces platelet CD40 ligand and thrombin generation in hypercholesterolemic patients

BACKGROUND Soluble CD40L (sCD40L), a substance that maximally reflects in vivo platelet activation, is increased in patients with hypercholesterolemia. We investigated the relation between sCD40L and platelet CD4OL in hypercholesterolemic patients before and after a short-term treatment with atorvastatin. METHODS AND RESULTS Collagen-induced platelet CD40L and plasma levels of sCD40L and prothrombin fragment F1+2, a marker of thrombin generation, were investigated in 30 hypercholesterolemic patients and 20 healthy subjects. Hypercholesterolemic patients were then randomized to either diet (n=15; group A) or atorvastatin 10 mg/d (group B); the aforementioned variables were measured at baseline and after 3 days of treatment. Compared with referents, hypercholesterolemic patients showed higher values of platelet CD40L (P<0.005), sCD40L (P<0.005), and F1+2 (P<0.003). Platelet CD40L was significantly correlated with sCD40L (P<0.001), and the latter was significantly correlated with F1+2 (P<0.001). The intervention trial showed no changes in group A but a significant decrease in platelet CD40L (P<0.01), sCD40L (P<0.002), and F1+2 (P<0.03) in group B. In vitro studies demonstrated that cholesterol enhanced platelet CD40L and CD40L-mediated clotting activation by human monocytes; also, atorvastatin dose-dependently inhibited platelet CD40L expression and clotting activation by CD40L-stimulated monocytes. CONCLUSIONS This study shows that, in hypercholesterolemia, platelet overexpression of CD40L may account for enhanced plasma levels of sCD40L and F1+2. Atorvastatin exerts a direct antithrombotic effect via inhibition of platelet CD40L and CD40L-mediated thrombin generation, independently of its cholesterol-lowering effect.

gp91phox-Dependent Expression of Platelet CD40 Ligand

Background—CD40 ligand (CD40L) expression on platelets is mediated by agonists, but the underlying mechanism is still unclear. Methods and Results—CD40L expression was measured in platelets from healthy subjects both with and without the addition of antioxidants or a phospholipase A2 (PLA2) inhibitor and in platelets from 2 patients with an inherited deficiency of gp91phox. Immunoprecipitation analysis was also performed to determine whether normal platelets showed gp91phox expression. Unlike catalase and mannitol, superoxide dismutase inhibited agonist-induced platelet CD40L expression in healthy subjects. Immunoprecipitation analysis also showed that platelets from healthy subjects expressed gp91phox. In 2 male patients with inherited gp91phox deficiency, collagen-, thrombin-, and arachidonic acid-stimulated platelets showed an almost complete absence of superoxide anion (O2−) and CD40L expression. Incubation of platelets from healthy subjects with a PLA2 inhibitor almost completely prevented agonist-induced O2− and CD40L expression. Conclusions—These data provide the first evidence that platelet CD40L expression occurs via arachidonic acid–mediated gp91phox activation.

Hereditary deficiency of gp91(phox) is associated with enhanced arterial dilatation: results of a multicenter study.

Background— NADPH oxidase is believed to modulate arterial tone, but its role in humans is still unclear. The objective of this study was to evaluate whether NADPH oxidase is involved in flow-mediated arterial dilation (FMD). Methods and Results— Twenty-five patients with hereditary deficiency of gp91phox, the catalytic core of NADPH oxidase, (X-CGD), 25 healthy subjects, and 25 obese patients matched for sex and age were recruited. FMD, platelet gp91phox, serum levels of nitrite and nitrate as markers of nitric oxide generation, oxidized low-density lipoprotein, and urinary excretion of isoprostanes as markers of oxidative stress were determined. Platelet gp91phox expression was downregulated in X-CGD patients (1.0±0.8 mean fluorescence; P<0.001) and upregulated in obese patients (4.1±2.2 mean fluorescence; P=0.01) compared with healthy subjects (2.9±1.7 mean fluorescence). Urinary excretion of isoprostanes was reduced in X-CGD patients (41.7±33.3 pg/mg creatinine; P=0.04) and increased in obese patients (154.4±91 pg/mg creatinine; P<0.001) compared with healthy subjects (69.5±52.4 pg/mg creatinine). Obese patients had higher serum oxidized low-density lipoprotein than healthy subjects (35.3±6.7 versus 24.8±9.8 U/L; P<0.001) and X-CGD patients (28.5±7.2 U/L; P<0.001). X-CGD patients had significantly higher FMD (14.7±5.9%) compared with healthy subjects (7.9±2.5%; P<0.001); obese patients had lower FMD (5.3±3.0%; P=0.028) compared with healthy subjects. Serum nitrite and nitrate levels were significantly higher in patients with X-CGD (36.0±10.8 μmol/L; P=0.016) and lower in obese patients (9.3±11.0 μmol/L; P=0.001) compared with healthy subjects (27.1±19.1 μmol/L). Serum nitrite and nitrate levels significantly correlated with FMD (Rs=0.403, P<0.001) and platelet gp91phox (Rs=−0.515, P<0.001). FMD inversely correlated with platelet gp91phox (Rs=−0.502, P<0.001) and isoprostanes (Rs=−0.513, P<0.001). Conclusion— This study provides the first evidence that, in humans, gp91phox is implicated in the modulation of arterial tone.

Polyphenols enhance platelet nitric oxide by inhibiting protein kinase C-dependent NADPH oxidase activation: effect on platelet recruitment

Several studies demonstrated an inverse association between polyphenol intake and cardiovascular events. Platelet recruitment is an important phase of platelet activation at the site of vascular injury, but it has never been investigated whether polyphenols influence platelet recruitment. The aim of the study was to analyze in vitro whether two polyphenols, quercetin and catechin, were able to affect platelet recruitment. Platelet recruitment was reduced by NO donors and by NADPH oxidase inhibitors and was enhanced by L‐NAME, an inhibitor of NO synthase. Quercetin and catechin, but not single polyphenol, significantly inhibited platelet recruitment in a concentration‐dependent fashion. The formation of superoxide anion was significantly inhibited in platelets incubated with quercetin and catechin but was unaffected by a single polyphenol. Incubation of platelets with quercetin and catechin resulted in inhibition of PKC and NADPH oxidase activation. Treatment of platelets with quercetin and catechin resulted in an increase of NO and also down‐regulated the expression of GpIIb/IIIa glycoprotein. This study shows that the polyphenols quercetin and catechin synergistically act in reducing platelet recruitment via inhibition of PKC‐dependent NADPH oxidase activation. This effect, resulting in NO‐mediated platelet glycoprotein GpIIb/IIIa down‐regulation, could provide a novel mechanism through which polyphenols reduce cardiovascular disease.—Pignatelli, P., Di Santo, S., Buchetti, B., Sanguigni, V., Brunelli, A., Violi, F. Polyphenols enhance platelet nitric oxide by inhibiting protein kinase C‐dependent NADPH oxidase activation: effect on platelet recruitment. FASEB J. 20, 1082–1089 (2006)

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.

Atorvastatin Inhibits gp91phox Circulating Levels in Patients With Hypercholesterolemia

Objective—The inhibition of oxidative stress is among the most relevant pleiotropic effects of statins. The mechanism by which statins exert their antioxidant effect in vivo is still undefined. NADPH oxidase is among the most important sources of reactive oxygen species involved in atherosclerotic disease. Methods/Results—We developed an ELISA to evaluate serum levels of soluble-gp91phox, the catalytic core of phagocyte NADPH oxidase. In a cross-sectional study performed in 30 hypercholesterolemic patients and in 20 controls, serum soluble-gp91phox and urinary isoprostane, a marker of oxidative stress, were measured. The 2 variables were also measured in hypercholesterolemic patients, randomized to diet (n=15), or diet plus atorvastatin (10 mg daily, n=15) and followed for 30 days. Compared to controls, hypercholesterolemic patients had higher and significantly correlated (R=0.71; P<0.001) serum soluble-gp91phox (P<0.001) and urinary isoprostanes (P<0.001). After follow-up, the statin-allocated group showed a significant reduction of soluble-gp91phox (−33%, P<0.01), that paralleled that of isoprostanes (−37%, P<0.01) and cholesterol (−25%, P<0.01). The diet-allocated group showed only a weak reduction of cholesterol. Conclusion—Our study demonstrates that statins exert an antioxidant effect via inhibition of soluble gp91phox expression.

Oxidative Stress Is Associated With Arterial Dysfunction and Enhanced Intima-Media Thickness in Children With Hypercholesterolemia: The Potential Role of Nicotinamide-Adenine Dinucleotide Phosphate Oxidase

BACKGROUND. Endothelial dysfunction and intima-media thickness are precocious manifestations of hypercholesterolemia, but the mechanism is unclear. OBJECTIVE. The aim of the study was to analyze the interplay among endothelial dysfunction, intima-media thickness, and oxidative stress in children with hypercholesterolemia. METHODS. We performed a cross-sectional study comparing flow-mediated dilation, intima-media thickness, lipid profile, urinary isoprostanes as markers of oxidative stress, and platelet expression of gp91phox, the catalytic unit of nicotinamide-adenine dinucleotide phosphate oxidase, in a population of 50 children with hypercholesterolemia (mean age ± SD: 10.0 ± 3.7 years) and 50 children without hypercholesterolemia (mean age: 9.2 ± 3.5 years). Four children with hereditary deficiency of gp91phox were studied also. RESULTS. Children with hypercholesterolemia had reduced flow-mediated dilation (mean ± SD: 6.2 ± 2.4 vs 9.2 ± 2.5%) and enhanced intima-media thickness (0.45 ± 0.07 vs 0.40 ± 0.06 mm), urinary isoprostanes (86.9 ± 51.6 vs 45.9 ± 25.6 pg/mg creatinine), and gp91phox platelet expression (4.4 ± 3.8 vs 2.0 ± 1.7 mean fluorescence) compared with control subjects. At bivariate analysis, flow-mediated dilation was correlated with low-density lipoprotein cholesterol, intima-media thickness, urinary isoprostanes, and platelet gp91phox. Stepwise multiple linear regression analysis showed that, in children with hypercholesterolemia, flow-mediated dilation and intima-media thickness were significantly associated with low-density lipoprotein cholesterol and urinary isoprostanes; also, gp91phox platelet expression was an independent predictor of urinary isoprostanes. Children with gp91phox hereditary deficiency showed downregulation of platelet gp91phox and reduced urinary excretion of isoprostanes. CONCLUSIONS. The study suggests that gp91phox-mediated oxidative stress may have a pathogenic role in the anatomic and functional changes of the arterial wall occurring in children with premature atherosclerosis.

Metformin decreases platelet superoxide anion production in diabetic patients

Patients with type 2 diabetes mellitus are usually treated with oral antidiabetic agents but it is still not known whether these drugs have antioxidant effects in humans.

LDL are oxidatively modified by platelets via GP91phox and accumulate in human monocytes

Oxidative stress‐mediated LDL modification has a key role in initiation of the atherosclerotic process. Platelets produce reactive oxidant species (ROS) upon stimulation with agonist, but it is uncertain whether they are able to oxidatively modify LDL. Human platelets taken from healthy subjects were incubated with LDL, then stimulated with collagen. Compared with unstimulated platelets, collagen‐stimulated platelets induced LDL modification as shown by enhanced conjugated dienes and lysophosphatidylcho‐line formation, electrophoretic mobility, Apo B‐100 degradation, and monocyte LDL uptake. Activated platelets also induced a marked reduction of vitamin Ε contained in LDL. A significant inhibition of LDL oxidation was observed in platelets treated with arachi‐donyl trifluomethyl ketone (AACOCF3), an inhibitor of phospolipase A2. The experiments reported above were also conducted in patients with hereditary deficiency of gp91phox, the central core of NADPH oxidase, and in patients with hypercholesterolemia. Platelets from gp91 phox‐deficient patients produced a small amount of ROS and weakly modified LDL. Conversely, platelets from hypercholesterolemic patients showed enhanced ROS formation and oxidized LDL more than platelets from healthy subjects. This study provides evidence that platelets modify LDL via NADPH oxidase‐mediated oxidative stress, a phenomenon that could be dependent on arachidonic acid activation. This finding suggests a role for platelets in favoring LDL accumulation within atherosclerotic plaque.—Carnevale, R., Pignatelli, P., Lenti, L., Buchetti B., Sanguigni, V., Di Santo, S., Violi, F. LDL are oxidatively modified by platelets via GP91phox and accumulate in human monocytes. FASEB J. 21, 927–934 (2007)

Reduced Atherosclerotic Burden in Subjects With Genetically Determined Low Oxidative Stress

Objective—NADPH oxidase, one of the most important enzymes producing reactive oxidant species, is suggested to play a role in experimental atherosclerosis, but its role in human atherosclerosis is still unclear. We hypothesized that a reduced activity of NADPH oxidase might be linked to a reduced atherosclerotic burden. Methods and Results—Thirty-one women carriers of hereditary deficiency of NOX2, the catalytic subunit of NADPH oxidase, were matched for sex and age with 31 controls and 31 obese women. Flow-mediated dilation and intima-media thickness, 2 surrogate markers of atherosclerosis, serum activity of NOX2, urinary isoprostanes, serum levels of nitrite/nitrate, and platelet production of isoprostanes and nitrite/nitrate were determined. Compared with controls (5.7±3.0% and 0.60±0.11 mm), carriers of NOX2 deficiency had higher flow-mediated dilation (9.2±5.0%; P<0.001) and lower intima-media thickness (0.50±0.11 mm; P=0.002), whereas obese women had lower flow-mediated dilation (3.2±2.1%; P=0.007) and higher intima-media thickness (0.71±0.15 mm; P<0.001). Compared with controls, carriers of NOX2 deficiency had lower urinary isoprostanes (132.6±87.3 versus 82.3±46.0 pg/mg creatinine; P=0.007) and serum NOX2 activity (24.9±19.3 versus 12.8±11.9 pg/mL; P=0.004) and higher serum nitrite/nitrate (23.8±7.6 versus 30.5±6.3 µmol/L; P<0.001), whereas obese women had higher urinary isoprostanes (132.6±87.3 versus 182.2±84.6 pg/mg creatinine; P=0.008) and serum NOX2 activity (24.9±19.3 versus 36.1±18.6 pg/mL; P=0.008) and lower serum nitrite/nitrate (23.8±7.6 versus 12.6±4.2 µmol/L; P<0.001). Flow-mediated dilation correlated with intima-media thickness (r=–0.433; P<0.001), serum NOX2 activity (r=–325; P<0.001), and urinary isoprostanes (r=–0.314; P=0.002). Ex vivo study showed that, compared with controls, platelets from carriers of NOX2 deficiency had lower isoprostanes (P<0.001) and higher nitrite/nitrate (P<0.001), whereas platelets from obese women had higher isoprostanes (P<0.001) and lower nitrite/nitrate (P=0.013). Conclusion—The study shows reduced atherosclerotic burden in carriers of NOX2 deficiency, suggesting that oxidative stress generated by this enzymatic pathway is implicated in human atherosclerosis.