Alessandra Meneguzzi

NCX-4016 (NO-Aspirin) Inhibits Lipopolysaccharide-Induced Tissue Factor Expression In Vivo Role of Nitric Oxide

Background—NCX-4016 is an acetylsalicylic acid (ASA) derivative containing a nitric oxide–releasing moiety. Compared with ASA, NCX-4016 has a broader spectrum of antithrombotic and antiinflammatory activities. We hypothesized that NCX-4016 might inhibit in vivo lipopolysaccharide (LPS)-induced expression of tissue factor (TF). Methods and Results—Rats were administered 90 mg/kg NCX-4016 orally for 5 days. Placebo, 50 mg/kg ASA, and 80 mg/kg isosorbide-5-mononitrate (ISMN) were used in control groups. On day 5, rats were injected intraperitoneally with 100 &mgr;g/kg LPS and killed 6 hours later. The expression of TF in monocytes was measured by flow cytometry and Western blot analysis. Reverse transcriptase–polymerase chain reaction was performed to assess expression of TF and cyclooxygenase-2 (COX-2) genes. Plasma concentrations of interleukin-1&bgr; and tumor necrosis factor-&agr; were measured. Urine samples were collected to evaluate the excretion of the thromboxane metabolite 11-dehydro-thromboxane (TX)B2. Gastric mucosa was inspected. LPS injection was followed by synthesis TF and COX-2 mRNAs in circulating monocytes, which were blunted by NCX-4016 but not by ASA or ISMN. Both NCX-4016 and ISMN reduced TF expression on surface of circulating monocyte. LPS increased the excretion 11-dehydro-TXB2, and this was prevented by NCX-4016 and ASA. Unlike ASA, NCX-4016 reduced plasma interleukin-1&bgr; and tumor necrosis factor-&agr;. In addition, NCX-4016 almost completely prevented mucosal damage, whereas ASA increased the extension of gastric lesions in LPS-injected rats. Conclusions—NCX-4016 prevents monocyte TF expression; this is accompanied by inhibition of TX and cytokine biosynthesis. These additive effects of nitric oxide release and COX inhibition may help explain efficacy and tolerability of NCX-4016.

NCX4016 (NO-Aspirin) has multiple inhibitory effects in LPS-stimulated human monocytes.

NCX4016 (2 acetoxy‐benzoate 2‐(2‐nitroxymethyl)‐phenyl ester, NicOx S.A., France) is an anti‐thrombotic agent, chemically related to acetylsalicylic acid (ASA) and able to release NO. We tested the effects of NCX4016 and ASA on the release of the thromboxane (TX) A2 metabolite TXB2, tumour necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), expression and activity of tissue factor (TF) in stimulated, adherent human monocytes. Both ASA and NCX4016 1 – 1000 μmol l−1 dose‐dependently reduced TXB2 concentration, measured by RIA in the supernatant of 10 μg ml−1 LPS‐stimulated cells. NCX4016 activity was comparable to that of equimolar ASA when incubation lasted 6 h (NCX4016 30 μmol l−1: −86.0±10.1%, NCX4016 300 μmol l−1: −92.2±9.0%, ASA 30 μmol l−1: −92.3±7.5%, ASA 300 μmol l−1: −97.3±1.0%, n=6, M±s.d.). Most of the activity of NCX4016 up to 100 μmol l−1 was prevented by 10 μmol l−1 ODQ, inhibitor of cyclic GMP. NCX4016 100 – 300 μmol l−1 reduced TNF‐α (NCX4016 300 μmol l−1=−77.2±19.9%, n=6) and IL‐6 (NCX4016 300 μmol l−1: −61.9±15.2%, n=6) in LPS stimulated monocytes while ASA had no significant effects. TF activity (NCX4016 300 μmol l−1: 53.7±39.9%, n=4) and immunoreactive TF (NCX4016 300 μmol l−1: −93.9±7.9%, n=7), measured in the supernatant of stimulated cells, were also dose‐dependently inhibited by NCX4016 but not by ASA. The present results indicate that NCX4016 inhibits TXA2 generation as well as cytokine release and TF in human monocytes partly via NO‐dependent mechanisms. NCX4016 may have a favourable profile of activities in the clinical setting of athero‐thrombosis.

Increased protein nitration in mitochondrial diseases: evidence for vessel wall involvement.

Mitochondrial diseases (MD) are heterogeneous disorders because of impairment of respiratory chain function leading to oxidative stress. We hypothesized that in MD the vascular endothelium may be affected by increased oxidative/nitrative stress causing a reduction of nitric oxide availability. We therefore, investigated the pathobiology of vasculature in MD patients by assaying the presence of 3-nitrotyrosine in muscle biopsies followed by the proteomic identification of proteins which undergo tyrosine nitration. We then measured the flow-mediated vasodilatation as a proof of altered nitric oxide generation/bioactivity. Here, we show that 3-nitrotyrosine staining is specifically located in the small vessels of muscle tissue and that the reaction is stronger and more evident in a significant percentage of vessels from MD patients as compared with controls. Eleven specific proteins which are nitrated under pathological conditions were identified; most of them are involved in energy metabolism and are located mainly in mitochondria. In MD patients the flow-mediated vasodilatation was reduced whereas baseline arterial diameters, blood flow velocity and endothelium-independent vasodilatation were similar to controls. The present results provide evidence that in MD the vessel wall is a target of increased oxidative/nitrative stress.

Antiplatelet Agents Inhibit the Generation of Platelet-Derived Microparticles

Platelet microparticles (PMPs) contribute to thrombogenesis but the effects of antiplatelet drugs on PMPs generation is undefined. The present study investigated the cellular events regulating PMPs shedding, testing in vitro platelet agonists and inhibitors. Platelet-rich plasma from healthy subjects was stimulated with arachidonic acid (AA), U46619, collagen type-I (10 and 1.5 μg/mL), epinephrine, ADP or TRAP-6 and pre-incubated with acetylsalicylic acid (ASA, 100 and 10 μmol/L), SQ-29,548, apyrase, PSB-0739, or eptifibatide. PMPs were detected by flow-cytometry using CD61 and annexin-V as fluorescent markers. Platelet agonists induced annexin V-positive PMPs shedding. The strongest response was to high concentration collagen. ADP-triggered PMPs shedding was dose-independent. ASA reduced PMPs induced by AA- (645, 347–2946 vs. 3061, 446–4901 PMPs/μL; median ad range, n = 9, P < 0.001), collagen 10 μg/mL (5317, 2027–15935 vs. 10252, 4187–46316 PMPs/μL; n = 13, P < 0.001), collagen 1.5 μg/mL (1078, 528–2820 vs. 1465, 582–5948 PMPs/μL; n = 21, P < 0.001) and TRAP-6 (2008, 1621–2495 vs. 2840, 2404–3031 PMPs/μL; n = 3, P < 0.01) but did not affect the response to epinephrine or ADP. The ADP scavenger apyrase reduced PMPs induced by U46619 (1256, 395–2908 vs. 3045, 1119–5494 PMPs/μL, n = 6, P < 0.05), collagen 1.5 μg/mL (1006, 780–1309 vs. 2422, 1839–3494 PMPs/μL, n = 3, P < 0.01) and TRAP-6 (904, 761–1224 vs. 2840, 2404–3031 PMPs/μL, n = 3, P < 0.01). The TP receptor antagonist SQ-29,548 and the P2Y12 receptor antagonist PSB-0739 markedly inhibited PMPs induced by low doses of collagen. Except for high-dose collagen, eptifibatide abolished agonist-induced PMPs release. Both TXA2 generation and ADP secretion are required as amplifiers of PMP shedding. The crucial role of the fibrinogen receptor and the collagen receptor in PMPs generation, independently of platelet aggregation, was identified.

Collagen I and III mRNA gene expression and cell growth potential of skin fibroblasts in patients with essential hypertension

Objectives Despite the claimed disregulation of extracellular matrix synthesis and the increased proliferation rate of different cell types in experimental models of hypertension, very few data are available on collagen synthesis and the proliferation rate of fibroblasts in essential hypertensive patients. Design We measured collagen I, collagen III, histone H3 mRNA gene expression, collagen protein concentration and thymidine incorporation in fibroblasts from 17 essential hypertensive patients (EH) and 13 healthy normotensive control subjects (NC). Methods A Northern blot analysis was performed on fibroblasts in culture obtained from skin biopsies. Collagen protein concentration and DNA synthesis were measured by means of incorporation of tritiated proline and tritiated thymidine, respectively. Results In cultivated fibroblasts from hypertensives, the expression of collagen III mRNA after addition of fetal calf serum was significantly increased in comparison with that of normotensive-derived cells. After addition of fetal calf serum, collagen protein was statistically increased in cultures from EH patients as compared to NC. In hypertensives, the expression of histone H3 mRNA as well as tritiated thymidine incorporation were both increased as compared to normotensives. Conclusions Our data suggest that cultivated fibroblasts from essential hypertensive patients are characterized by an increased expression of type III collagen mRNA and collagen protein synthesis in response to fetal serum, as compared to normotensive-derived cells. Cells from hypertensives are characterized by an increased rate of proliferation after addition of fetal serum, as ascertained by increased thymidine incorporation and increased histone H3 mRNA gene expression, as compared to normotensive-derived cells. This phenotype could be genetically determined and may have an important role in the pathogenesis of essential hypertension.

Genetic grafting of membrane-acting peptides to the cytotoxin dianthin augments its ability to de-stabilize lipid bilayers and enhances its cytotoxic potential as the component of transferrin-toxin conjugates.

Three chimeric proteins were obtained by fusing together the dianthin gene and DNA fragments encoding for the following membrane‐acting peptides: the N‐terminus of protein G of the vesicular stomatitis virus (KFT25), the N terminus of the HA2 hemagglutinin of influenza virus (pHA2), and a membrane‐acting peptide (pJVE). Chimeric dianthins (KFT25DIA, pHA2DIA and pJVEDIA) retained full enzymatic activity in cell‐free assays and showed increased ability to induce pH‐dependent calcein release from large unilamellar vesicles (LUVs). pHA2DIA and pJVEDIA also showed faster kinetics of interaction with LUVs, while KFT25DIA and pHA2DIA displayed a reduced cytotoxicity as compared to wild‐type dianthin. Conjugates made by chemically cross‐linking KFT25DIA or pJVEDIA and human transferrin (Tfn) showed greater cell‐killing efficiency than conjugates of Tfn and wild‐type dianthin. As a consequence, by fusion of membrane‐acting peptides to the dianthin sequence the specificity factor (i.e., the ratio between non‐specific and specific toxicity) of Tfn‐KFT25DIA, Tfn‐pHA2DIA and Tfn‐pJVEDIA was increased with respect to that of Tfn‐based conjugates made with wild‐type dianthin. Taken together, our results suggest that genetic fusion of membrane‐acting peptides to enzymatic cytotoxins results in the acquisition of new physico‐chemical properties exploitable for designing new recombinant cytotoxins and to tackle cell‐intoxication mechanisms. Int. J. Cancer 86:582–589, 2000.

Differential regulation of TNF receptors in maternal leukocytes is associated with severe preterm preeclampsia

Abstract We tested the hypothesis that maternal peripheral blood leukocytes contribute to elevated levels of soluble TNF receptors (sTNFR) in preeclampsia (PE) with concomitant intrauterine growth restriction (IUGR). TNFR1 and TNFR2 were evaluated in a cross-sectional study comparing preeclamptic (n = 15) with or without IUGR versus normotensive pregnant women (PREG, n = 30), and non-pregnant controls (Con; n = 20). Plasma levels of sTNFR1 were higher in PE (1675.0 ± 227.1 pg/mL) compared with PREG (1035.0 ± 101.1 pg/mL) and Con (589.3 ± 82.67 pg/mL), with the highest values observed in PE with IUGR (2624.0 ± 421.4 pg/mL; n = 6). Plasma sTNFR2 was higher during pregnancy (PE: 1836.0 ± 198.7 pg/mL; PREG: 1697.0 ± 95.0 pg/mL) compared with Con (598.3 ± 82.7 pg/mL). Urinary levels of sTNFR1 and sTNFR2 were higher in PE and PREG compared with the Con group. Abundance of TNFR1 mRNA in peripheral blood leukocytes was strongly correlated with plasma levels of sTNFR1 in PE. However, TNFR2 mRNA accumulation in leukocytes did not correlate with sTNFR2 plasma levels. The level of sTNFR1 in plasma was correlated with body weight of the newborn (r = −0.56). The data suggest that maternal leukocytes contribute to sTNFR1 levels in plasma in association with decreasing newborn weight and PE with concomitant IUGR.

Endothelial dysfunction and increased oxidative stress in mitochondrial diseases

MDs (mitochondrial diseases) are a clinically heterogeneous group of disorders characterized by impairment of the respiratory chain function with altered oxidative phosphorylation. We tested the hypothesis that the function of vascular endothelium is affected by increased oxidative stress in MDs. A total of 12 patients with MDs and pair-matched controls were studied. Endothelial function was assessed by measuring FMD (flow-mediated vasodilation) of brachial and common femoral arteries. The test was repeated after vitamin C (500 mg, twice a day) and E (400 mg, once a day) supplementation for 30 days and 90 days after vitamin withdrawal. FMD was reduced in patients compared with controls [AUC/τ (time-averaged area under the curve) for the brachial artery, 1.05±0.24 compared with 4.19±0.59% respectively, P<0.001; AUC/τ for the femoral artery, 0.98±0.19 compared with 2.36±0.29% respectively, P=0.001; values are means±S.E.M.] and correlated (brachial artery) with plasma lactate (r=-0.63, P<0.01). Urinary 8-iso-PGF2α (8-iso-prostaglandin F2α) was higher in patients than controls (505.6±85.9 compared with 302.5±38.7 pg/mg of creatinine; P<0.05) and correlated with plasma lactate (r=0.70, P<0.05). Immunohistochemical analysis showed 8-iso-PGF2α staining in MD-affected striated muscle cells and in blood vessels in muscle biopsies of patients. Antioxidant vitamins transiently restored FMD in patients [ΔAUC/τ (change in AUC/τ) for the brachial artery, +1.38±0.49%, P<0.05; ΔAUC/τ for the femoral artery, +0.98±0.24%, P<0.01] but had no effect on FMD in controls (brachial artery, -1.3±0.63%; and common femoral artery, -0.58±0.30%), thus abolishing the differences between patients and controls. The results of the present study indicate that oxidative stress is increased and is, at least partly, responsible for endothelial dysfunction in MDs.

Calcium-Dependent Src Phosphorylation and Reactive Oxygen Species Generation Are Implicated in the Activation of Human Platelet Induced by Thromboxane A2 Analogs

The thromboxane (TX) A2 elicits TP-dependent different platelet responses. Low amounts activate Src kinases and the Rho–Rho kinase pathway independently of integrin αIIbβ3 and ADP secretion and synergize with epinephrine to induce aggregation. Aim of the present study was to investigate the role Src kinases and the interplay with calcium signals in reactive oxygen species (ROS) generation in the activatory pathways engaged by TXA2 in human platelets. All the experiments were performed in vitro or ex vivo. Washed platelets were stimulated with 50–1000 nM U46619 and/or 10 μM epinephrine in the presence of acetylsalicylic acid and the ADP scavenger apyrase. The effects of the ROS scavenger EUK-134, NADPH oxidase (NOX) inhibitor apocynin, Src kinase inhibitor PP2 and calcium chelator BAPTA were tested. Intracellular calcium and ROS generation were measured. Platelet rich plasma from patients treated with dasatinib was used to confirm the data obtained in vitro. We observed that 50 nM U46619 plus epinephrine increase intracellular calcium similarly to 1000 nM U46619. ROS generation was blunted by the NOX inhibitor apocynin. BAPTA inhibited ROS generation in resting and activated platelets. Phosphorylation of Src and MLC proteins were not significantly affected by antioxidants agents. BAPTA and antioxidants reduced P-Selectin expression, activation of integrin αIIbβ3and platelet aggregation. TXA2-induced increase in intracellular calcium is required for Src phosphorylation and ROS generation. NADPH oxidase is the source of ROS in TX stimulated platelets. The proposed model helps explain why an incomplete inhibition of TP receptor results in residual platelet activation, and define new targets for antiplatelet treatment.

Reduced Variability to Aspirin Antiplatelet Effect by the Coadministration of Statins in High‐Risk Patients for Cardiovascular Disease

We studied the influence of cardiovascular (CV) risk factors, previous CV events, and cotreatments with preventive medicines, on residual platelet thromboxane (TX)B2 production in 182 patients chronically treated with enteric coated (EC)‐aspirin (100 mg/day). The response to aspirin was also verified by assessing arachidonic acid‐induced platelet aggregation and urinary 11‐dehydro‐TXB2 levels. Residual serum TXB2 levels exceeded the upper limit value for an adequate aspirin response in 14% of individuals. This phenomenon was detected at 12 hours after dosing with aspirin. The coadministration of statins (mostly atorvastatin) was an independent predictor of residual serum TXB2 levels, and the percentage of patients with enhanced values was significantly lower in statin users vs. nonusers. We provide evidence in vitro that atorvastatin reduced residual TXB2 generation by increasing the extent of acetylation of platelet COX‐1 by aspirin. In conclusion, the coadministration of statins may counter the mechanisms associated with reduced bioavailability of aspirin detected in some individuals with CV disease.