Alessio Metere

Effect of therapeutic inhibition of TNF on circulating endothelial progenitor cells in patients with rheumatoid arthritis.

Endothelial dysfunction has been detected in RA patients and seems to be reversed by control of inflammation. Low circulating endothelial progenitor cells (EPCs) have been described in many conditions associated with increased cardiovascular risk, including RA. The aim of this study was to investigate the effect of inhibition of TNF on EPCs in RA patients. Seventeen patients with moderate-severe RA and 12 sex and age-matched controls were evaluated. Endothelial biomarkers were tested at baseline and after 3 months. EPCs were identified from peripheral blood mononuclear cells by cytofluorimetry using anti-CD34 and anti-vascular endothelial growth factor-receptor 2. Asymmetric dimethylarginine (ADMA) was tested by ELISA and flow-mediated dilatation (FMD) by ultrasonography. Circulating EPCs were significantly lower in RA patients than in controls (P = 0.001). After 3 months EPCs increased significantly (P = 0.0006) while ADMA levels significantly decreased (P = 0.001). An inverse correlation between mean increase in EPCs number and mean decrease of DAS28 after treatment was observed (r = −0.56, P = 0.04). EPCs inversely correlated with ADMA (r = −0.41, P = 0.022). No improvement of FMD was detected. Short-term treatment with anti-TNF was able to increase circulating EPCs concurrently with a proportional decrease of disease activity suggesting that therapeutic intervention aimed at suppressing the inflammatory process might positively affect the endothelial function.

Serum Levels of Asymmetric Dimethylarginine and Apelin as Potential Markers of Vascular Endothelial Dysfunction in Early Rheumatoid Arthritis

Objectives. Impaired endothelial function represents the early stage of atherosclerosis, which is typically associated with systemic inflammatory diseases like rheumatoid arthritis (RA). As modulators of endothelial nitric oxide synthase expression, asymmetric-dimethylarginine (ADMA) and apelin might be measured in the blood of RA patients to detect early atherosclerotic changes. We conducted a prospective, case-control study to investigate serum ADMA and apelin profiles of patients with early-stage RA (ERA) before and after disease-modifying antirheumatic drug (DMARD) therapy. Methods. We enrolled 20 consecutively diagnosed, treatment-naïve patients with ERA and 20 matched healthy controls. Serum ADMA and apelin levels and the 28-joint disease activity scores (DAS28) were assessed before and after 12 months of DMARDs treatment. All patients underwent ultrasonographic assessment for intima-media tickness (IMT) evaluation. Results. In the ERA group, ADMA serum levels were significantly higher than controls at baseline (P = 0.007) and significantly decreased after treatment (P = 0.012 versus controls). Baseline serum apelin levels were significantly decreased in this group (P = 0.0001 versus controls), but they were not significantly altered by treatment. IMT did not show significant changes. Conclusions. ERA is associated with alterations of serum ADMA and apelin levels, which might be used as biomarkers to detect early endothelial dysfunction in these patients.

Oxidative stress and defective platelet apoptosis in naïve patients with Kawasaki disease

Kawasaki disease (KD) is a rare and often undiagnosed disease, at least in the western countries. It is characterized by an inflammatory acute febrile vasculitis of medium sized arteries with a propensity to damage the coronary arteries. It normally occurs in the early childhood and the diagnosis is based on clinical symptoms. During the progression of the disease thrombocytosis is usually detected. This can exert a pathogenetic role in the cardiovascular complications occurring in KD. In the present work peripheral blood plasma and platelets from twelve naïve patients with KD were analyzed in order to detect possible pathogenetic determinants or progression markers. Morphological, biochemical and flow cytometrical methods have been used. With respect to age-matched healthy donors, we found an increase of platelet activation markers, i.e. degranulation, phosphatidylserine (PS) externalization and leukocyte-red cell-platelet aggregates. Some significant alterations that could represent suitable diagnostic determinants have also been detected in patient plasma: (i) decreased antioxidant power, (ii) decreased levels of asymmetric dymethylarginine (ADMA), a naturally occurring chemical interfering with the production of nitric oxide, and (iii) increased levels of soluble P-Selectin and soluble annexin V. Since PS externalizing platelets are known to exert a pro-coagulant activity, our data suggest the hypothesis that increased risk of vascular complications in KD could depend on platelet stimulation and defective apoptosis probably related to nitrosative stress.

Does Oxidative Stress Play a Critical Role in Cardiovascular Complications of Kawasaki Disease

The aim of the present work was to evaluate the contribution of the different reactive oxidizing species to systemic oxidative stress in the whole blood of patients with Kawasaki disease (KD). This is a rare generalized systemic vasculitis typical of the early childhood characterized by inflammation and endothelial dysfunction with a high risk for cardiovascular fatal events. We found that, compared to age-matched healthy donors, blood from KD patients showed increased production of oxygen- and nitrogen-derived species as detected by electron paramagnetic resonance (EPR) spin probing with the cyclic hydroxylamine 1-hydroxy-3-carboxy-pyrrolidine. The (•)NO pathway involvement was also confirmed by the decreased concentrations of the endogenous (•)NO synthase inhibitor asymmetric dimethyl-arginine and the increased amounts of 3-nitrotyrosine in plasma. Further, increased plasma yields of the proinflammatory enzyme myeloperoxidase were also observed. The appearance of circulating red blood cell alterations typically associated with oxidative imbalance and premature aging (e.g., decrease of total thiol content, glycophorin A, and CD47 expression, as well as increase of phosphatidylserine externalization) has also been detected. Collectively, our observations lead to hypothesize that the simultaneous oxidative and nitrative stress occurrence in the blood of KD patients may play a pathogenetic role in the cardiovascular complications often associated with this rare disease.

Red Blood Cells as a Model to Differentiate between Direct and Indirect Oxidation Pathways of Peroxynitrite

Red blood cells are the major physiological scavengers of reactive nitrogen species and have been proposed as real-time biomarkers of some vascular-related diseases. This chapter proposes that the erythrocyte is a suitable cell model for studying the modifications induced by peroxynitrite. Peroxynitrite decays both extra- and intracellularly as a function of cell density and CO(2) concentration, inducing the appearance of distinct cellular biomarkers, as well as the modulation of signaling and metabolism. Intracellular oxidations are due mostly to direct reactions of peroxynitrite with hemoglobin but also lead to the appearance of apoptotic biomarkers. Surface/membrane oxidations are due principally to indirect radical reactions generated by CO(2)-catalyzed peroxynitrite homolysis.

Decrease of Asymmetric Dimethyl Arginine After Anti-TNF Therapy in Patients with Rheumatoid Arthritis

Postmarketing Phase IV

Peroxynitrite signaling in human erythrocytes: synergistic role of hemoglobin oxidation and band 3 tyrosine phosphorylation.

Peroxynitrite crosses the red blood cell (RBC) membrane and reacts with hemoglobin (Hb) producing mainly metHb, which is reduced back to ferrousHb by NADH- and NADPH-dependent reductases. Peroxynitrite also induces band 3 (B3) tyrosine phosphorylation, a signaling pathway believed to activate glucose metabolism. This study was aimed to decipher the relationship between these two peroxynitrite-dependent processes. Peroxynitrite induced a burst of the hexose monophosphate shunt (HMS), revealed by NMR studies, and a burst of the glycolytic pathway, measured by lactate production. The HMS plays a prominent role in membrane signaling, as demonstrated by B3 phosphotyrosine inhibition by the glycolytic pathway inhibitor 2-deoxy-glucose (2DG) and activation by dehydroepiandrosterone (DHEA), an inhibitor of HMS. Peroxynitrite-induced B3 tyrosine phosphorylation was paralleled by the inhibition of membrane-associated phosphotyrosine phosphatase (PTP) activity, which was protected by 2DG but not DHEA. Interestingly, heme poisoning with CO inhibited peroxynitrite-dependent Hb oxidation and lactate production but did not affect PTP down regulation. These results suggest two distinct and concurrent effects of peroxynitrite: one mediated by Hb which, likely in its oxidized state, binds more strongly to B3, and another mediated by PTP-dependent B3 phosphorylation. Both effects are directed towards a surge in glucose utilization.

L‐NAME reverses quinolinic acid‐induced toxicity in rat corticostriatal slices: Involvement of src family kinases

Quinolinic acid (QA) is an endogenous excitotoxin acting on N‐methyl‐d‐aspartate receptors (NMDARs) that leads to the pathologic and neurochemical features similar to those observed in Huntingtons disease (HD). The mechanism of QA toxicity also involves free radicals formation and oxidative stress. NMDARs are particularly vulnerable to the action of reactive oxygen species (ROS) and reactive nitrogen species (RNS) that can act as modulators of the activity of protein tyrosine kinases (PTKs) and phosphotyrosine phosphatases (PTPs). Because QA is able to activate neuronal nitric oxide synthase (nNOS) as well as to stimulate the NMDARs, we evaluated the effect of Nω‐Nitro‐l‐arginine‐methyl ester (l‐NAME), a selective nNOS inhibitor, on QA‐induced neurotoxicity in rat corticostriatal slices. In electrophysiologic experiments we observed that slice perfusion with QA induced a strong reduction of field potential (FP) amplitude, followed by a partial recovery at the end of the QA washout. In the presence of l‐NAME the recovery of FP amplitude was significantly increased with respect to QA alone. In synaptosomes, prepared from corticostriatal slices after the electrophysiologic recordings, we observed that l‐NAME pre‐incubation reversed the QA‐mediated inhibitory effects on protein tyrosine phosphorylation pattern, c‐src, lyn, and fyn kinase activities and tyrosine phosphorylation of NMDAR subunit NR2B, whereas the PTP activity was not recovered in the presence of l‐NAME. These findings suggest that NO plays a key role in the molecular mechanisms of QA‐mediated excitotoxicity in experimental model of HD.

Quinolinic acid modulates the activity of src family kinases in rat striatum: in vivo and in vitro studies.

Quinolinic acid (QA) has been shown to evoke neurotoxic events via NMDA receptor (NMDAR) overactivation and oxidative stress. NMDARs are particularly vulnerable to free radicals, which can modulate protein tyrosine kinase (PTK) and phosphotyrosine phosphatase (PTP) activities. The src family of tyrosine kinases are associated with the NMDAR complex and regulate NMDA channel function. Because QA is an NMDAR agonist as well as a pro‐oxidant agent, we investigated whether it may affect the activity of PTKs and PTPs in vivo and in vitro. In synaptosomes prepared from striata dissected 15 min, 30 min or 15 days after bilateral injection of QA we observed modulation of the phosphotyrosine pattern; a significant decrease in PTP activity; and a sustained increase in c‐src and lyn activity at 15 and 30 min after treatment with QA, followed by a decrease 2 weeks later. Striatal synaptosomes treated in vitro with QA showed time‐ and dose‐dependent modulation of c‐src and lyn kinase activities. Moreover, the nitric oxide synthase inhibitor NG‐nitro‐l‐arginine‐methyl ester, the NMDAR antagonist d‐2‐amino‐5‐phosphonovaleric acid and pyruvate suppressed the QA‐induced modulation of c‐src activity. These findings suggest a novel feature of QA in regulating src kinase activity through the formation of reactive radical species and/or NMDAR overactivation.