Search

Hormones and cytokines are known to regulate cellular functions in the testes. These biomolecules induce a broad spectrum of effects on various level of spermatogenesis, and among them is the modulation of cell junction restructuring between Sertoli cells and germ cells in the seminiferous epithelium. Cytokines and androgens are closely related, and both correct testicular development and the maintenance of spermatogenesis depend on their function. Cytokines also play a crucial role in the immune testicular system, activating and directing leucocytes across the endothelial barrier to the inflammatory site, as well as in increasing their adhesion to the vascular wall. The purpose of this review is to revise the most recent findings on molecular mechanisms that play a key role in male sexual function, focusing on three specific molecular patterns, namely, cytokines, miRNAs, and endothelial progenitor cells. Numerous reports on the interactions between the immune and endocrine systems can be found in the literature. However, there is not yet a multi-approach review of the literature underlying the role between molecular patterns and testicular and sexual function.

Oxidative stress may be defined as an imbalance between reactive oxygen species (ROS) and the antioxidant system to counteract or detoxify these potentially damaging molecules. This phenomenon is a common feature of many human disorders, such as cardiovascular disease. Many of the risk factors, including smoking, hypertension, hypercholesterolemia, diabetes, and obesity, are associated with an increased risk of developing cardiovascular disease, involving an elevated oxidative stress burden (either due to enhanced ROS production or decreased antioxidant protection). There are many therapeutic options to treat oxidative stress-associated cardiovascular diseases. Numerous studies have focused on the utility of antioxidant supplementation. However, whether antioxidant supplementation has any preventive and/or therapeutic value in cardiovascular pathology is still a matter of debate. In this review, we provide a detailed description of oxidative stress biomarkers in several cardiovascular risk factors. We also discuss the clinical implications of the supplementation with several classes of antioxidants, and their potential role for protecting against cardiovascular risk factors.

NOX2 has a key role for cellular production of reactive oxidant species (ROS) and although the mechanism of its activation is well known, little is known about its regulation. Metallo-proteinases (MMPs) regulate numerous protein activities both in physiological and pathological conditions but their interplay with NOX2 and ROS formation is still unclear. We performed experimental studies in human platelets and polymorphonuclear leukocytes (PMNs) to investigate the interplay of MMP2 with NOX2 activity. In collagen-stimulated platelets and in PMA-stimulated PMNs from healthy subjects, an immediate burst of ROS was detected at 10 min to then decline at 20 min. Coincidentally, sNOX2-dp, a split-off product of NOX2, increased and peaked at 10 min. ROS production was persistent whereas sNOX2dp is not released in cells treated with MMP2 inhibitor compared to other MMPs inhibitors. Western blot analysis showed the highest MMP2 expression on the cell membrane 10 min after stimulation. Moreover, the co-immunoprecipitation assay confirms the interaction between MMP2 and NOX2 that formed an active immuno-complex. Treating cells with NOX2ds-tat, an inhibitor of NADPH oxidase, significantly reduced ROS formation, sNOX2-dp, MMP2 expression and MMP2-NOX2-complex, which were all restored if cells were added with H 2 O 2 . The study provides the first evidence that MMP2 has a key role in blunting platelet NOX2 activity and eventually ROS formation., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: The formation of reactive oxygen species (ROS) contributes to the pathogenesis and progression of several diseases. Polyphenols have been shown to be beneficial against ROS. The aim of this study was to evaluate the effects of a natural antioxidant ice cream on oxidative stress, vascular function, and physical performance., Methods: In this controlled, single-blind, crossover study, 14 healthy individuals were randomized to consume 100 g of either antioxidant ice cream containing dark cocoa powder and hazelnut and green tea extracts or milk chocolate ice cream (control ice cream). Participants were studied at baseline and 2 h after ingesting ice cream. Serum polyphenols, antioxidant status (ferric-reducing ability of plasma [FRAP]), nitric oxide (NOx) bioavailability, markers of oxidative stress (determination of reactive oxygen metabolites [d-ROMs] and hydrogen peroxide [H 2 O 2 ]), endothelium function (flow-mediated dilation [FMD] and reactive hyperemia index [RHI]), and exercise tolerance (stress test) were assessed, and the double product was measured., Results: Serum polyphenols (P < 0.001), NOx (P < 0.001), FRAP (P < 0.005), FMD (P < 0.001), and RHI (P < 0.05) increased significantly, oxidative stress decreased (d-Roms, P < 0.001; H 2 O 2 , P < 0.001), and the double product (P < 0.001) was improved only after antioxidant ice cream ingestion. No changes were found after control ice cream ingestion., Conclusions: To our knowledge, this is the first study to demonstrate that a natural ice cream rich in polyphenols acutely improved vascular function and physical performance in healthy individuals through a reduction in oxidative stress., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: In vitro study showed that NADPH oxidase (NOx), the most important enzyme producing reactive oxygen species (ROS), plays a role in the process of platelet activation. However, it is unclear if changes in its activity affect platelet activation in vivo., Methods and Results: In vivo and ex vivo experiments assessing platelet activation were investigated in healthy subjects, obese patients, and subjects with different low rates of NOx2 activity, namely X-linked chronic granulomatous disease (X-CGD) patients and X-CGD carriers. We included 27 X-CGD patients, 31 women carriers of hereditary deficiency of NOx2, 31 obese women, and 62 healthy subjects matched for sex and age. Plasma levels of soluble sCD40 L (sCD40L) and soluble P (sP)-selectin, 2 markers of in vivo platelet activation, were reduced in X-CGD patients (sCD40L=-55%; sP-selectin=-51%, P<0.001) and in X-CGD carriers (sCD40L=-41%; sP-selectin=-57%, P<0.001) compared with respective controls. Conversely, obese women, who disclosed NOx2 upregulation, had significantly higher plasma levels of sCD40L (+47%, P<0.001) and sP-selectin (+70%, P<0.001) compared with controls. Ex vivo study showed platelet isoprostane downexpression and enhanced platelet NO generation in both X-CGD patients and X-CGD carriers compared with controls; opposite findings were observed in obese patients. Correlation analysis showed that platelet NOx2 regulation was directly associated with plasma levels of sCD40L (R=0.336, P<0.001) and sP-selectin (R=0.441; P<0.001)., Conclusions: The study provides the first evidence that in vivo platelet activation is significantly and directly associated with NOx2 activity. Platelet NOx2 may be a novel target for platelet activation inhibition., (© 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

NADPH oxidase is known to modulate the arterial tone, but the role of its specific subunits is still unclear. The objective of this study was to compare the role of p47 and gp91phox (NOX2) on artery dilatation. We conducted a multicenter study enrolling 30 patients with chronic granulomatous disease (CGD) (25 with NOX2 deficiency and 5 with p47(phox) deficiency) and 30 healthy subjects (HS), matched for gender and age, in whom flow-mediated dilation (FMD), serum activity of NOX2 (soluble NOX2-derived peptide [sNOX2-dp]), urinary isoprostanes (8-iso-PGF2α), and platelet production of isoprostanes and NOX2 were determined. Compared to HS, patients with CGD had significantly higher FMD and lower sNOX2-dp and 8-iso-PGF2α levels. Compared to patients with NOX2 deficiency and HS, patients with p47(phox) hereditary deficiency had intermediate FMD and oxidative stress, that is, higher and lower FMD and lower and higher isoprostanes compared to HS and patients with NOX2 deficiency, respectively. In agreement with this finding, an ex vivo study showed higher inhibition of NOX2 activity and lower isoprostane formation in platelets from patients with NOX2 deficiency compared to platelets from ones with p47(phox) deficiency. Our observations lead to the hypothesis that oxidants are implicated in artery vasoconstriction.

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)., Conclusions: 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.

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(phox) hereditary deficiency had normal thromboxane A(2) formation but marked 8-iso-PGF2α reduction compared with controls. In normal platelets incubated with a gp91(phox) inhibitor or with SQ29548, a thromboxane A(2)/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 Ca(2+) and activating gpIIb/IIIa; a further increase in platelet recruitment was detected by platelet coincubation with l-NAME, an inhibitor of NO synthase., Conclusions: This study provides the first evidence that platelet 8-iso-PGF2α maximally derives from gp91(phox) activation and contributes to platelet recruitment via activation of gpIIb/IIIa.

Background: Interventional treatment with atorvastatin lowered the circulating levels of the catalytic core of NADPH oxidase, namely sgp91(phox), but the underlying mechanism is still undefined., Aim: To test the hypothesis that the inhibitory effect on oxidative stress, induced by Atorvastatin, could be mediated by adiponectin., Methods and Results: We compared 36 patients with polygenic hypercholesterolemia and 18 healthy subjects. Patients were randomized to either a low-fat diet (Group A) or low-fat diet plus atorvastatin 10 mg/day (Group B) for 30 days. Lower serum adiponectin levels and higher lipid profile, gp91(phox) serum levels, urinary isoprostanes, platelet oxygen free radicals, characterized patients. After 30 days of treatment, group B showed higher levels of adiponectin which is inversely correlated to reduced levels of sgp91(phox), urinary isoprostanes and platelet oxygen free radicals (p<0.001). In in vitro model, adiponectin dosages between 5 and 10 ng/ml inhibited p47(phox) translocation to gp91(phox) and soluble gp91(phox) cleavage indicating its ability in inhibiting the assembly of NADPH oxidase subunits on cell membrane and in turn the enzymatic system activation., Conclusion: This study provides the first evidence that in patients higher APN serum levels are associated with gp91(phox) down-regulation. APN-mediated gp91(phox) reduction could be one of the mechanisms involved in atorvastatin's antioxidant effect., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

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-gp91(phox), the catalytic core of phagocyte NADPH oxidase. In a cross-sectional study performed in 30 hypercholesterolemic patients and in 20 controls, serum soluble-gp91(phox) 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-gp91(phox) (P<0.001) and urinary isoprostanes (P<0.001). After follow-up, the statin-allocated group showed a significant reduction of soluble-gp91(phox) (-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., Conclusions: Our study demonstrates that statins exert an antioxidant effect via inhibition of soluble gp91(phox) expression.

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 gp91(phox), the catalytic core of NADPH oxidase, (X-CGD), 25 healthy subjects, and 25 obese patients matched for sex and age were recruited. FMD, platelet gp91(phox), 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 gp91(phox) 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 micromol/L; P=0.016) and lower in obese patients (9.3+/-11.0 micromol/L; P=0.001) compared with healthy subjects (27.1+/-19.1 micromol/L). Serum nitrite and nitrate levels significantly correlated with FMD (R(s)=0.403, P<0.001) and platelet gp91(phox) (R(s)=-0.515, P<0.001). FMD inversely correlated with platelet gp91(phox) (R(s)=-0.502, P<0.001) and isoprostanes (R(s)=-0.513, P<0.001)., Conclusions: This study provides the first evidence that, in humans, gp91(phox) is implicated in the modulation of arterial tone.

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 gp91(phox), 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 gp91(phox) 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 gp91(phox) 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 gp91(phox). 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, gp91(phox) platelet expression was an independent predictor of urinary isoprostanes. Children with gp91(phox) hereditary deficiency showed downregulation of platelet gp91(phox) and reduced urinary excretion of isoprostanes., Conclusions: The study suggests that gp91(phox)-mediated oxidative stress may have a pathogenic role in the anatomic and functional changes of the arterial wall occurring in children with premature atherosclerosis.

Aims: Statins inhibit oxidative stress, but the interplay between cholesterol lowering and antioxidant vitamins is still unclear. Aims of the study were to assess if statins inhibit oxidative stress independently from cholesterol lowering, to assess the behaviour of vitamin E simultaneously with the changes of oxidative stress, to determine in vitro if atorvastatin was able to directly influence platelet-mediated LDL oxidation and vitamin E consumption., Methods and Results: In 30 hypercholesterolaemic patients (HC) and 20 healthy subjects (HS), urinary isoprostanes and plasma vitamin E were determined. The HC were randomized to diet or diet plus atorvastatin 10 mg/day. Compared with HS, HC had higher isoprostanes and lower vitamin E levels. The statin-allocated group showed a reduction of isoprostanes after only 3 days (-18.8%, P < 0.01); after 30 days, a stronger reduction of isoprostanes was noted (-37.1%, P < 0.01) whereas an increase of vitamin E (+42%, P < 0.01) and a reduction of cholesterol (-24.9%, P < 0.01) were observed. The diet-allocated group showed a weak decrease of cholesterol after 30 days. In vitro study showed that atorvastatin dose-dependently inhibited platelet-mediated LDL oxidation and isoprostane formation with a mechanism involving NADPH-oxidase., Conclusion: The study provides the first evidence that atorvastatin exerts an early antioxidant effect that could contribute to enhancing circulating vitamin E.

Objectives: We speculated that in patients with hypercholesterolemia CD40L overexpression could depend on low-density lipoprotein (LDL)-induced enhanced intraplatelet formation of O(2)*(-) and statin could reduce platelet CD40L via interference with platelet O(2)*(-) production., Background: CD40L is a protein with inflammatory and thrombotic properties. CD40L is upregulated in platelets from hypercholesterolemic (HC) patients but the underlying mechanism is unclear., Methods: Collagen-induced platelet CD40L and platelet O(2)*(-) expression were investigated in 40 HC patients and 40 healthy subjects. HC patients were then randomized to either a diet (n = 20) (group A) or atorvastatin 10 mg day (n = 20) (group B); the above variables were measured at baseline and after 3 and 30 days of treatment. O(2)*(-) and CD40L were also measured in vitro in LDL-treated platelets with or without nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor or atorvastatin added., Results: Compared with controls, HC patients showed higher values of platelet CD40L (P < 0.001) and O(2)*(-) (P < 0.001). Platelet CD40L was significantly correlated with O(2)*(-) (P < 0.001). The interventional trial showed no changes in group A and a significant and parallel decrease in platelet CD40L (P < 0.001) and O(2)*(-) (P < 0.001) in group B. In vitro studies demonstrated that LDL-induced platelet CD40L and GP IIb/IIIa (PAC1 binding) activation via the NADPH oxidase pathway. CD40L upregulation was counteracted by atorvastatin in a dose-dependent fashion., Conclusions: This study suggests that in patients with hypercholesterolemia platelet CD40L is upregulated via NADPH oxidase-dependent O(2)*(-) generation. Atorvastatin downregulated CD40L with an oxidative stress-mediated mechanism likely involving platelet NADPH oxidase, an effect that seemed to be independent of its cholesterol-lowering action.

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 lysophosphatidylcholine formation, electrophoretic mobility, Apo B-100 degradation, and monocyte LDL uptake. Activated platelets also induced a marked reduction of vitamin E contained in LDL. A significant inhibition of LDL oxidation was observed in platelets treated with arachidonyl trifluomethyl ketone (AACOCF3), an inhibitor of phospholipase 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.

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.

Upon stimulation with agonists, platelets express CD40 ligand (CD40L), a transmembrane protein implicated in the initiation and progression of atherosclerotic disease. We have recently discovered that oxidative stress plays a major role in platelet CD40L expression. In this study, we sought to determine whether vitamin C, a known antioxidant, is able to influence platelet CD40L expression. In vitro experiments were done by stimulating platelets with collagen in the presence or absence of vitamin C (50-100 microM) or vehicle as control. An in vivo study was done in 10 healthy subjects who were randomized to intravenous infusion of placebo or 1 g vitamin C for 45 min in a crossover design. At the end of infusion platelet CD40L and O2- were measured. The in vitro study demonstrated that vitamin C dose dependently inhibited platelet CD40L expression without affecting agonist-induced platelet aggregation. In subjects treated with placebo no changes of platelet CD40L and O2- were observed; conversely, vitamin C infusion caused a significant and parallel decrease of platelet O2- (-70%, P < 0.001) and CD40L (-68%, P < 0.001). Platelet aggregation was not modified by either treatment. This study suggests that water-soluble antioxidants, which scavenge superoxide radicals, may reduce platelet CD40L expression.

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.

Objectives: We tested the hypothesis that CD40 ligand (CD40L) induces a prothrombotic state by enhancing oxidative stress., Background: Patients with hypercholesterolemia show an ongoing prothrombotic state, but the underlying mechanism is still unclear., Methods: Circulating levels of the soluble form of CD40L (sCD40L), prothrombin fragment (F1+2, a marker of thrombin generation), and 8-hydroxy-2'-deoxyguanosine (8-OHdG, a marker of oxidative stress) were measured in 40 patients with hypercholesterolemia and in 20 age- and gender-matched healthy subjects., Results: Patients with hypercholesterolemia showed significantly higher levels of sCD40L (p <0.005), 8-OHdG (p <0.005), and prothrombin F1+2 (p <0.005), as compared with control subjects. Soluble CD40L significantly correlated with 8-OHdG (r=0.85, p <0.0001) and prothrombin F1+2 (r=0.83, p <0.0001); a significant correlation between 8-OHdG and prothrombin F1+2 was also observed (r=0.64, p <0.0001). An in vitro study demonstrated that CD40L-stimulated monocytes from patients with hypercholesterolemia expressed more tissue factor (TF) and prothrombin F1+2 than monocytes from controls; co-incubation of monocytes with either an inhibitor of NADPH oxidase or an inhibitor of phosphatidylinositol-3-kinase significantly reduced CD40L-mediated clotting activation. A marked inhibition of CD40L-mediated clotting activation was also observed in two male patients with hereditary deficiency of gp91 phox, the central core of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Finally, we demonstrated that CD40L-mediated clotting activation was significantly inhibited by vitamin C, a known antioxidant., Conclusions: This study indicates that in patients with hypercholesterolemia, CD40L over-expresses TF and increases the thrombin generation rate by an oxidative stress-mediated mechanism that requires the activation of NADPH oxidase.

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 (O(2)(-)) and CD40L expression. Incubation of platelets from healthy subjects with a PLA2 inhibitor almost completely prevented agonist-induced O(2)(-) and CD40L expression., Conclusions: These data provide the first evidence that platelet CD40L expression occurs via arachidonic acid-mediated gp91phox activation.

Background: Enhanced oxidative stress has been observed in hypertension, but the underlying mechanism has not been fully clarified., Objective: To study the relationship between oxygen free radicals and hypertension, using platelets as a tool to measure the cellular production of superoxide anion (O2)., Design: Forty patients with hypertension were allocated randomly to groups to receive either irbesartan, an inhibitor of angiotensin II type 1 (AT1) receptors (n = 20), or a diuretic (hydrochlorothiazide) (n = 20). In each patient, collagen-induced production of O2 by platelets was studied before and after 4 weeks of treatment. Forty sex- and age-matched healthy individuals were studied as controls., Methods: Platelet-produced O2 was measured using lucigenin chemiluminescence and hydroethidine cytofluorimetric analysis., Results: Compared with healthy individuals, patients with hypertension showed a greater production of O2 by platelets (P < 0.001); there was no correlation between blood pressure and platelet O2 production. After treatment, no changes in platelet O2 formation were observed in patients receiving hydrochlorothiazide; conversely, those treated with irbesartan showed a significant (P < 0.001) decrease in platelet O2 production. At the end of the treatment, no differences in blood pressures were observed between the two groups. In-vitro incubation of platelets with angiotensin II elicited a significant increase in O2 (P < 0.001) that was dose-dependently inhibited by irbesartan and diphenylene iodonium, an inhibitor of NADPH oxidase., Conclusion: Patients with hypertension showed an enhanced formation of O2 in platelets that was not dependent on blood pressure but could be mediated by AT1 receptors via NADPH oxidase activation.

Carnitine is a physiological cellular constituent that favors intracellular fatty acid transport, whose role on platelet function and O(2) free radicals has not been fully investigated. The aim of this study was to seek whether carnitine interferes with arachidonic acid metabolism and platelet function. Carnitine (10-50 microM) was able to dose dependently inhibit arachidonic acid incorporation into platelet phospholipids and agonist-induced arachidonic acid release. Incubation of platelets with carnitine dose dependently inhibited collagen-induced platelet aggregation, thromboxane A(2) formation, and Ca(2+) mobilization, without affecting phospholipase A(2) activation. Furthermore, carnitine inhibited platelet superoxide anion (O(2)(-)) formation elicited by arachidonic acid and collagen. To explore the underlying mechanism, arachidonic acid-stimulated platelets were incubated with NADPH. This study showed an enhanced platelet O(2)(-) formation, suggesting a role for NADPH oxidase in arachidonic acid-mediated platelet O(2)(-) production. Incubation of platelets with carnitine significantly reduced arachidonic acid-mediated NADPH oxidase activation. Moreover, the activation of protein kinase C was inhibited by 50 microM carnitine. This study shows that carnitine inhibits arachidonic acid accumulation into platelet phospholipids and in turn platelet function and arachidonic acid release elicited by platelet agonists.

Objectives: The purpose of this study was to investigate the relationship between hypercholesterolemia and superoxide anion production., Background: Experimental studies demonstrated that hypercholesterolemia is associated with enhanced cellular superoxide anion (O2-) production. Aim of the study was to assess whether the same phenomenon occurs in humans., Methods: Lipid profile and platelet O2- production were measured in 28 patients with hypercholesterolemia, compared with 25 age- and sex-matched healthy subjects, and in 21 out of the 28 patients after 8-week treatment with 10 mg/day atorvastatin (a HMGCoA reductase inhibitor). In order to assess the mechanism by which LDL cholesterol interferes with platelet production of O2-, human platelets were incubated with LDL cholesterol in the presence of either an inhibitor of the phospholipaseA2 enzyme, AACOCF3, or an inhibitor of NADH/NADPH oxidases, DPI., Results: O2- platelet generation was significantly higher (p <0.001) and significantly related to LDL cholesterol (p <0.001 ) in patients as compared to controls. 8-week treatment with 10 mg/day atorvastatin significantly reduced both LDL cholesterol and O2- platelet production. This effect was partially related to the cholesterol-lowering, in that three days of treatment with atorvastatin significantly decreased platelet O2- production, while no significant change in LDL-cholesterol levels was observed. Platelets incubated with LDL cholesterol showed an increased O2- production, which was significantly inhibited by AACOCF3 (-78%) and DPI (-56%)., Conclusions: LDL cholesterol increases platelet O2- production by activating PLA2 and NADH/NADPH enzymes. Inhibition of platelet O2- release by atorvastatin is partially related to cholesterol lowering effect, suggesting that other mechanisms could be responsible for the antioxidant activity of the drug.

Background: 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., Methods: We studied 60 patients with type 2 diabetes mellitus, divided into three groups on the basis of hypoglycaemic treatment (Group A: metformin, Group B: glibenclamide, Group C: diet). All patients were followed for at least 1 year. The three subgroups had similar clinical characteristics. Twenty healthy subjects, of comparable sex and age, were enrolled as controls. In each subject, platelet production of superoxide anion (O(2)(-)) elicited by collagen, was determined by lucigenin assay., Results: Patients with diabetes had higher platelet O(2)(-) production than controls; no correlation was observed between blood glucose and platelet O(2)(-) production. Group A patients had platelet O(2)(-) production similar to that of healthy subjects but lower than Group B and Group C patients., Conclusion: The present findings suggest an in vivo antioxidant activity of metformin and warrant prospective studies to further explore this hypothesis., (Copyright 2002 John Wiley & Sons, Ltd.)

Previous study demonstrated that platelets undergoing anoxia-reoxygenation generate superoxide anion (O2-) and hydroxyl radical (OH ) which in turn contribute to activate arachidonic acid (AA) metabolism. However it has not been clarified if oxygen free radicals (OFRs) are also generated when platelets are aggregated by common agonists. We used two probes, i.e. lucigenin and salicylic acid (SA), to measure platelet release of O2- and OH(0), respectively. Among the agonists used, such as ADP, thrombin and collagen, the release of O2- and OH was observed mainly when platelets were stimulated with collagen. Such release was inhibited in platelets pre-treated by aspirin suggesting that AA metabolism was the main source of O2- and OH(0) formation. To further analyze this relationship, O2- and OH(0) formation was measured during AA-stimulated platelet aggregation (PA); we observed that O2- and OH(0) release were dependent upon AA concentration. Furthermore, we found that the incubation of platelets with AACOCF3, a potent inhibitor of cytosolic phospholipase A2, inhibited collagen-induced platelet O2- and OH(0) release. The incubation of platelets with salicylic acid or ascorbic acid, which blunt OH and O2- respectively, inhibited both collagen-induced platelet aggregation and AA-release. This study demonstrated that collagen-induced platelet aggregation is associated with O2- and OH formation, which is dependent upon AA release and metabolism.

Background: Literature concerning exercise-induced platelet activation in chronic stable angina is somewhat confusing. The reason lies in the type of exercise as well as in methodological problems. A powerful, recently introduced procedure to detect platelet activation is flow cytometry. Platelet response to activating factors is mediated by calcium uptake; however, calcium antagonist effect on platelet activity is still unclear., Hypothesis: The study was undertaken to investigate exercise-induced platelet activation before and after treatment with amlodipine in chronic stable angina., Methods: Twenty patients with chronic stable angina were entered into the study. Each subject underwent a symptom-limited cycloergometer stress test following a washout period of 2 weeks. Blood samples were collected before and immediately after exercise. All subjects were then randomized into two groups of 10 patients each, with Group 1 and Group 2 taking amlodipine 10 mg/day, and placebo for 4 weeks, respectively. They subsequently underwent a second exercise stress test, and blood samples were obtained before and immediately after exercise. Flow-cytometric evaluation of platelet activity was performed in order to recognize GMP-140 expression on platelet membrane., Results: Strenuous exercise induced a significant increase in platelet activation in all subjects prior to therapy. No significant differences were observed in platelet activity at rest between Groups 1 and 2, whereas a significant decrease in exercise-induced platelet activation was demonstrated in Group 1 compared with Group 2., Conclusion: Our data provide evidence of the favorable effect of amlodipine on exercise-induced platelet activation in patients affected by chronic stable angina.

In order to evaluate the effects of Italian brewed coffee (moka) on cholesterol and serum lipoproteins, a randomized double-blind 14-week clinical trial was performed. After a coffee-free period of four weeks, 49 subjects drank coffee, caffeinated and decaffeinated, for ten weeks. There was no evidence that the Italian method of brewing coffee affects serum lipoproteins since no statistically significant differences were found.

Antibiotic therapy has improved infective endocarditis prognosis. The observance of general rules to choose the more suitable antibiotic drugs, as regard to their effectiveness, pharmacodynamic peculiarities and use, is mandatory. If the infection is due to antibiotic resistant microorganisms, microbiological analyses to estimate the bactericidal effect of the antibiotics, must be carried out. Resistance to penicillins, oligopeptides and aminoglycosides makes endocarditis produced by Enterococcus spp difficult to treat. The identification of patients at risk for infective endocarditis after surgical and invasive instrumental procedures, allows to introduce antibiotic prophylaxis regimens which can reduce the probability of acquiring the disease.

In order to evaluate the effects of physical activity on seric lipoproteins and coagulation parameters, an 8-week clinical trial was performed. Fifteen healthy young subjects (average age 23 years) with no history of previous agonistic physical activity, entered the study. Each subject underwent a physical programme consisting of three times a week bicycle ergometer exercise with progressive increases in work rate by using stages of 3 min duration until the 85% of the predictive heart rate was reached. Each individual was subjected to four blood drawings according to the following schedule: at the beginning of the study, after 4 weeks, after 8 weeks (at the end of the programme) and 4 weeks after the interruption of training. As far as the seric lipoproteins are concerned, the following parameters were monitored: total cholesterol, HDL-C, LDL-C, VLDL-C, triglycerides, Apo-A1, Apo-B100, NEFA and phospholipids. On the other hand the following coagulation parameters were monitored: fibrinogen PT, aPtt, coagulation factors (II-XII), red cells, leucocytes, platelets, hemoglobin and hematocrit. From the analysis of the data, the following statistically significant results were observed: HDL-C increased by 14%, LDL-C decreased by 13%, Apo-A1 increased by 6%, fibrinogen increased by 31.7%, Ptt decreased by 3.7% and leucocytes increased by 15%. Four weeks after exercise was terminated, all monitored parameters turned into the basal range. Our data seem to demonstrate a positive effect of physical exercise on seric lipoproteins in the short period. Nevertheless they provide evidence of an hypercoagulability condition demonstrated by the important fibrinogen increase and the Ptt decrease.

In order to detect the presence of determining factors as predictors of progressive carotid atherosclerosis, the incidence of total serum cholesterol and fibrinogen elevation was evaluated in patients affected by coronary artery disease (CAD). 61 subjects with CAD (mean age 62 years) and significative lesions (> 50%) underwent periodic Echo-Doppler (Duplex scanning) of the supra aortic branches. Total serum cholesterol, HDL, LDL and fibrinogen were monitored, as well. A 24 month follow-up period was performed. Indicative of the progression of carotid atherosclerosis has been considered the presence of a stenosis degree over 20% than the initial one. In 14 of the 61 subjects who entered the study, there was evidence of progressive carotid atherosclerosis. The same patients showed higher levels of LDL cholesterol (130 +/- 36.3 vs 96.5 +/- 33.2) and Fibrinogen (398.3 +/- 59.4 vs 328 +/- 36.8) and lower levels of HDL cholesterol (27.2 +/- 4.2 vs 34.4 +/- 10.1). Our results confirm the importance of cholesterol and fibrinogen as determining risk factors, especially in patients with multiple vascular disease (coronary and carotid).

Silent myocardial ischemia (SMI) has been demonstrated in 2 to 5% of subjects in totally asymptomatic population, in 30% of patients with history of previous myocardial infarction and in 60 to 100% of patients with stable or unstable angina pectoris. In these patients, 60 to 80% of transient episodes of ischemia are silent and SMI is induced by daily activities and so can be registered during continuous ECG monitoring. The finding of SMI during an exercise testing or during ambulatory monitoring has an unfavourable prognostic significance both in apparently asymptomatic subjects and in patients suffering from stable or unstable angina pectoris or survivors to a myocardial infarction. Stress testing and Holter monitoring are the most used non invasive tests to detect SMI. The sensitivity and specificity of ergometer test can be improved by 201-Tl myocardial scintigraphy. Moreover, the ergometer test can be used as a provocative test to induce changes in regional wall kinesis and so these alterations can be evaluated by using echocardiogram and radioisotopic or contrast ventriculography. The echocardiogram allows to evaluate the presence of kinesis changes induced by stress test or by pharmacological stimulation with dipyridamole or dobutamine. SMI can be also detected through the study of metabolic alterations during cardiac catheterism.

The natural history of coronary artery disease (CAD) is often complicated by cerebrovascular accidents. The real incidence of atherosclerotic lesions of carotid arteries in coronary patients is not well established. In order to detect a high-risk group for stroke development, 184 patients affected by CAD, either partially symptomatic or asymptomatic for carotid artery stenosis, underwent Echo-Doppler ultrasonography (duplex scanning) of supra-aortic branches. Significant carotid stenosis (> 50%) was demonstrated in 51 subjects (27.7%); 21 subjects (41.2%) were partially symptomatic (dizziness, vertigo, lipothymia, etc), and 30 subjects (58.8%) were completely asymptomatic. The authors' data suggest that carotid disease can develop concurrently with coronary disease in a significant proportion of patients, even though completely asymptomatic. In order to obtain optimal long-term results, both coronary and carotid artery disease require appropriate evaluation and either medical or surgical management. For these reasons they recommend duplex scanning as a routine screening procedure in patients affected by CAD.

Three patients with total anomalous pulmonary venous drainage (TAPVD) were studied by real-time cross-sectional colour-flow Doppler echocardiography. Serial suprasternal, parasternal and subcostal scans were obtained. In all cases surgical or angiocardiographic confirmation was available. Two patients had supracardiac drainage (to the left vertical vein or to the right superior vena cava) and one patient had infracardiac drainage. An abnormal forward flow in the left innominate vein and vertical vein was visualized in those patients with supracardiac TAPVD. Abnormal venous flow was also imaged in one patient with mixed drainage. In the patient with infradiaphragmatic TAPVD characteristic flow signals were identified in the inferior vena cava (retrograde flow) and in the descending aorta and anomalous pulmonary venous channel (forward flow). In all patients the patterns of pulmonary venous flow allowed us to distinguish TAPVD from contiguous structures and to validate two-dimensional cross-sectional imaging.