A mass spectrometry-based workflow for the proteomic analysis of in vitro cultured cell subsets isolated by means of laser capture microdissection.

This paper describes a microproteomic workflow that is useful for simultaneously identifying and quantifying proteins from a minimal number of morphotypically heterogeneous cultured adherent cells. The analytical strategy makes use of laser capture microdissection, an effective means of harvesting pure cell populations, and label-free mass spectrometry. We optimised the workflow with particular reference to cell fixation which is crucial for successful laser-based microdissection and also downstream molecular studies. In addition, we defined the minimum number of cells to be isolated and analysed for satisfactory proteome coverage. To set up this workflow, we choose human monocyte-derived macrophages spontaneously differentiated in vitro. These cells, under our culture conditions, show distinct morphotypes, reminiscent of the heterogeneity observed in tissues in various homeostatic and pathological states, e.g. atherosclerosis. This optimised workflow may provide new insights into biology and pathology of heterogeneous cell in culture, particularly when other cell selection approaches are not suitable.

Human monocyte-derived macrophages spontaneously differentiated in vitro show distinct phenotypes.

Tissue macrophages are resident phagocytes that acquire specific phenotypes according to the microenvironment. Morphological and functional heterogeneity has been evidenced in different homeostatic and pathological conditions. Indeed, the nature of macrophage subsets may have either harmful or beneficial functions in disease progression/resolution. Therefore the possibility to pharmacologically manipulate heterogeneity represents a relevant challenge. Since human tissue macrophages are not easily obtained, various in vitro models are currently used that do not adequately reflect the heterogeneity and plasticity of tissue macrophages. We had previously reported that two dominant and distinct macrophage morphotypes co-exist in the same culture of human monocytes spontaneously differentiated for 7 days in autologous serum. The present study was aimed to the phenotypic characterization of these morphotypes, that is, round- and spindle-shaped. We observed that, besides substantial differences in cytoskeleton architecture, round monocyte-derived macrophages (MDMs) showed higher lipid content, increased macropinocytosis/efferocytosis capacity, and overexpression of CD163, interleukin (IL)-10, and transforming growth factor (TGF) ß2. Conversely, spindle MDMs exhibited enhanced respiratory burst and higher expression of the chemokine (C-C motif) ligands 18 and 24 (CCL18 and CCL24). Overall, round MDMs show functional traits reminiscent of the non-inflammatory and reparative M2 phenotype, whereas spindle MDMs exhibit a pro-inflammatory profile and express genes driving lymphocyte activation and eosinophil recruitment. MDMs obtained in the culture condition herein described represent a valuable model to disentangle and manipulate the functional heterogeneity of tissue macrophages that has been disclosed in scenarios spanning from inflammatory and wounding responses to atherosclerotic lesions.

Cytoskeletal architecture regulates cyclooxygenase-2 in human endothelial cells: autocrine modulation by prostacyclin.

Endothelium is a highly dynamic tissue that controls vascular homeostasis. This requires constant rearrangements of the shape or function of endothelial cells that cannot set aside the role of the cytoskeleton. The aim of this study was to determine the mechanisms by means of which cytoskeletal alterations induce cyclooxygenase-2 (Cox-2) expression in human endothelial cells using compounds that interfere with microtubule or actin architecture. Microtubule disruption by nocodazole markedly increased Cox-2 expression and activity, and provoked paracellular gap formation, a cardinal feature of endothelial barrier dysfunction. The Cox-2 metabolite prostacyclin down-regulated Cox-2 through an autocrine receptor-mediated mechanism, and partially prevented the disassembly of endothelial monolayers. There was also an interaction between microtubules and actin filaments in nocodazole-induced Cox-2 expression. Nocodazole provoked the dissolution of the F-actin cortical ring and stress fiber formation, increased actin glutathionylation, and concomitantly lowered intracellular levels of reduced glutathione. The restoration of glutathione levels by N-acetylcysteine opposed Cox-2 expression and preserved the integrity of endothelial monolayers. Among the signaling pathways connecting microtubule disruption with Cox-2 up-regulation, crucial roles are played by Src family kinase activation, serine/threonine phosphatase 2A inhibition, and the phosphorylation of mitogen activated protein kinase p38. Our findings provide a mechanistic insight into the observation that Cox-2 is induced in endothelial cells under cytoskeleton-perturbing conditions such as those occurring in the presence of atherogenic/inflammatory stimuli and oxidative stress. In this scenario, Cox-2 up-regulation by endothelia exposed to noxious conditions can be considered protective of the vasodilatory and anti-thrombotic properties of the vessel wall.

N-Acetylcysteine Inhibits Platelet Function through the Regeneration of the Non-Oxidative Form of Albumin.

N-acetylcysteine (NAC) is able to break down protein disulfides, generating free thiols. This mechanism occurs on mixed disulfides of albumin (HSA) to form mercaptoalbumin (HMA), the main antioxidant species in the plasma. Circulating HSA exists in two main forms: the reduced form (HMA), and the oxidized forms, whose predominant modification is cystenylation (HSA-Cys). Increased levels of oxidized HSA have been detected in several diseases associated with oxidative stress. This study showed that NAC inhibits platelet aggregation by restoring HMA. In addition, the regeneration of HMA by NAC inhibits platelet functions such as intracellular calcium mobilization, reactive oxygen species generation, arachidonic acid metabolites synthesis, and adhesion to the collagen matrix. In our conditions, the exposure of platelets to NAC did not increase GSH levels. However, the inhibition of platelet aggregation was also detected following treatment of platelet-rich plasma with GSH, which, similarly to NAC, reduced HSA-Cys levels. Furthermore, this study showed that cysteine, another compound able to restore HMA by reducing the HSA-Cys content, inhibited platelet aggregation to a similar extent as NAC. The results obtained in this study suggest a new mechanism by which NAC can modulate platelet activation and suggest its possible use as an antiplatelet drug in conditions associated with oxidative stress.

Cyclooxygenase-2 Glycosylation Is Affected by Peroxynitrite in Endothelial Cells: Impact on Enzyme Activity and Degradation.

The exposure of human endothelial cells to 3-morpholinosydnonimine (SIN-1) induced the expression of cyclooxygenase-2 (COX-2) in a dose- and time-dependent manner. Interestingly, after a prolonged incubation (>8 h) several proteoforms were visualized by Western blot, corresponding to different states of glycosylation of the protein. This effect was specific for SIN-1 that generates peroxynitrite and it was not detected with other nitric oxide-donors. Metabolic labeling experiments using 35S or cycloheximide suggested that the formation of hypoglycosylated COX-2 was dependent on de novo synthesis of the protein rather than the deglycosylation of the native protein. Moreover, SIN-1 reduced the activity of the hexokinase, the enzyme responsible for the first step of glycolysis. The hypoglycosylated COX-2 induced by SIN-1 showed a reduced capacity to generate prostaglandins and the activity was only partially recovered after immunoprecipitation. Finally, hypoglycosylated COX-2 showed a more rapid rate of degradation compared to COX-2 induced by IL-1α and an alteration in the localization with an accumulation mainly detected in the nuclear membrane. Our results have important implication to understand the effect of peroxynitrite on COX-2 expression and activity, and they may help to identify new pharmacological tools direct to increase COX-2 degradation or to inhibit its activity.

Anesthetic propofol enhances plasma gamma-tocopherol levels in patients undergoing cardiac surgery.

BACKGROUND: Propofol (2,6-diisopropylphenol) is an anesthetic drug with antioxidant and antiinflammatory properties, documented both in vitro and in experimental models of ischemia-reperfusion injury and septic shock. These properties have been related to the similarity of its chemical structure to that of endogenous tocopherols, which are phenol-containing radical scavengers. This study evaluated the effects of propofol on alpha- and gamma-tocopherol (alpha- and gamma-T) levels and on selected markers of oxidant-antioxidant and inflammatory status in patients undergoing cardiac surgery. METHODS: Patients were randomly assigned for anesthesia with either propofol (propofol group, n = 22) or sevoflurane (control group, n = 21). Plasma levels of alpha- and gamma-T, individual antioxidant capacity, malondialdehyde, and interleukin 10 were measured before, during, and after anesthesia. In addition, levels of the proinflammatory prostaglandin E2 as a marker of cyclooxygenase-2 activity and those of interleukin 10 were measured in whole blood cultured with bacterial lipopolysaccharide. RESULTS: Gamma-T levels increased significantly during surgery in propofol group (P < 0.0001 vs. control group). By contrast, alpha-T similarly decreased in both groups. Malondialdehyde and interleukin 10 increased markedly and individual antioxidant capacity decreased, without differences between groups. Prostaglandin E2 levels measured 24 h after anesthesia induction were significantly lower in the propofol than in the control group. In vitro studies highlighted the different capacity of gamma- and alpha-T to impair prostaglandin E2 synthesis by human monocytes challenged with bacterial lipopolysaccharide. CONCLUSIONS: The antiinflammatory properties of propofol that may be linked to its effect on gamma-T levels could be relevant in controlling the inflammatory response that accompanies tissue injury during reperfusion.

Paracrine up-regulation of monocyte cyclooxygenase-2 by platelets: role of transforming growth factor-beta1.

OBJECTIVE: To examine the role of platelets and platelet-derived products on cyclooxygenase-2 (Cox-2) induction in adherent monocytes and to address the signaling pathways involved. METHODS: Platelets and monocytes were obtained from peripheral blood of healthy donors. Adherent monocytes were co-cultured with autologous platelets or platelet releasates or exposed to mediators contained in platelet alpha-granules (either from platelet source or recombinant) for 4-24 h. Cox-2 protein and mRNA were determined by Western and RT-PCR analysis, respectively. Thromboxane B2 (TxB2) and prostaglandin E2 (PGE2) synthesis as index of Cox-2 activity, and levels of transforming growth factor-beta1 (TGF-beta1) in platelet releasates were measured by enzyme immunoassay (EIA). RESULTS: Activated platelets induce rapid and transient Cox-2 de novo synthesis in adherent monocytes. The effect is dependent upon the platelet number but not upon cell-cell contact. Platelet-induced Cox-2 was not affected by prevention of platelet TxA2 synthesis or microparticle formation but was blunted by inhibition of platelet alpha-granule secretion. TGF-beta1, either platelet-derived or recombinant (rTGF-beta1), induced Cox-2 expression and activity in adherent monocytes at concentrations within the range of those detected in releasates from activated platelets; this effect was not shared by recombinant platelet-derived growth factor (rPDGFBB). The time course of Cox-2 induction by TGF-beta1 in monocytes was identical to that observed with platelet releasates. Moreover, TGF-beta1 receptor blockade completely abolished platelet-induced Cox-2 expression. p38 MAPK activation represents a common transduction pathway through which activated platelets and rTGF-beta1 induce Cox-2 in monocytes. CONCLUSION: These findings suggest that TGF-beta1 released by activated platelets has a pivotal role in Cox-2 induction in monocytes and further supports the key role of platelets in the inflammatory and reparative responses.

Indobufen inhibits tissue factor in human monocytes through a thromboxane-mediated mechanism.

OBJECTIVE: To assess whether indobufen, a reversible inhibitor of platelet cyclooxygenase (Cox) activity, affects tissue factor (TF) in human monocytes and to investigate the relationship between Cox-derived products and TF. METHODS: TF was evaluated in isolated adherent monocytes, both resting and lipopolysaccharide (LPS)-stimulated, in terms of procoagulant activity, protein, and mRNA levels. The expression of TF surface antigen was determined in LPS-stimulated whole blood monocytes by flow cytometry. The levels of the stable thromboxane A2 (TxA2) metabolite, TxB2, and of prostaglandin E2 (PGE2) were measured in monocyte supernatant by immunoenzymatic techniques. Cox-1 and Cox-2 protein level, tyrosine phosphorylation, and mitogen-activated protein kinase (MAP-kinase) activation were determined by Western blot analysis. RESULTS: Indobufen prevents TF expression and activity both in isolated and in whole blood monocytes. Reduction of TxA2 synthesis, coupled with a lack of effect on PGE2 levels and prevention of ERK1/2 phosphorylation are highlighted as the mechanisms through which indobufen negatively affects TF. CONCLUSIONS: Data show that indobufen down-regulates TF in monocytes. This novel activity, coupled with the antiplatelet effect of the drug, may add benefit for its use in the management of atherothrombosis.

High-density lipoproteins attenuate interleukin-6 production in endothelial cells exposed to pro-inflammatory stimuli.

The purpose of the present study was to investigate the ability of high-density lipoproteins (HDL) to attenuate endothelial dysfunction, by assessing down-regulation of cytokine-induced interleukin-6 (IL-6) production in cultured endothelial cells, and measuring plasma IL-6 levels in three groups of healthy individuals with low, average, or high plasma HDL-cholesterol. Human plasma HDL caused a concentration-dependent inhibition of TNFalpha-induced IL-6 production in human endothelial cells (by 58.5+/-1.5% at 2 mg of HDL-protein/ml). Reconstituted HDL made with apolipoprotein A-I (apoA-I) and phosphatidylcholine were as effective as plasma HDL, while lipid-free apoA-I or phosphatidylcholine liposomes had no effect. HDL attenuated IL-6 mRNA levels, an effect which occurs through inhibition of p38 MAP kinase. The median plasma IL-6 concentration was significantly higher in subjects with low HDL-cholesterol (2.54 pg/ml) compared with those with average or high HDL-cholesterol (1.31 pg/ml and 1.47 pg/ml, respectively). When all subjects were considered together, a lower HDL-cholesterol was the strongest independent predictor of higher IL-6 (F=25.38, P<0.001). By inhibiting IL-6 production and lowering plasma IL-6 concentration, HDL may limit the pro-atherogenic effects of both acute and chronic inflammatory states, of which IL-6 is a key orchestrator.

Diversity and similarity in signaling events leading to rapid Cox-2 induction by tumor necrosis factor-alpha and phorbol ester in human endothelial cells.

OBJECTIVE: This study examines whether cyclooxygenase 2 (Cox-2) synthesis in human endothelial cells involves different signaling pathways when induced by the proinflammatory cytokine tumor necrosis factor-alpha (TNF alpha) or by the tumor and angiogenic promoter phorbol ester (PMA). Moreover, the hypothesis that reactive oxygen species (ROS) and an altered redox status within the cell are fundamental steps for Cox-2 synthesis is verified. METHODS: Human endothelial cells isolated from umbilical vein (HUVEC) were exposed to PMA and TNF alpha and Cox-2 protein and mRNA levels were evaluated by Western blot and Real-Time Quantitative Reverse Transcription-PCR analysis. Prostaglandin E(2) (PGE(2)) and 6-keto prostaglandin F(1 alpha) (6-keto-PGF(1 alpha)) levels were measured in cell medium as an index of Cox-2 activity. Intracellular ROS formation was detected by flow cytometry in HUVEC loaded with the oxidant-sensitive 2',7'-dichlorofluorescein diacetate (DCFH-DA) and by nitroblue tetrazolium (NBT) reduction. Reduced and oxidized glutathione (GSH and GSSG) were measured by HPLC. RESULTS: Data show that TNF alpha and PMA signal for early Cox-2 induction through distinct pathways. PMA-induced Cox-2 expression involves a small GTPase-dependent pathway acting via tyrosine kinase, activation of protein kinase C (PKC) and of the mitogen-activated protein kinase (MAPK) ERK1/2. Conversely, MAPK p38 is critical for Cox-2 induction by TNF alpha. Of interest, intracellular ROS generation and consequent GSH/GSSG ratio reduction represents a common step through which PMA and TNF alpha signal for early Cox-2 induction. In addition, we provide evidence that phosphatidylinositol 3 (PI3)-kinase activation plays a regulatory role for Cox-2 synthesis in HUVEC. CONCLUSION: Cox-2 represents a critical link among vascular homeostasis, inflammatory response, angiogenesis and tumor growth. The finding that two independent pathways and an overlapping upstream event signal for Cox-2 induction in HUVEC may be of relevance to develop strategies aimed at selectively interfering with Cox-2 regulating pathways.

Reactive oxygen species mediate cyclooxygenase-2 induction during monocyte to macrophage differentiation: critical role of NADPH oxidase.

OBJECTIVE: The objective of this study was to explore the relationship between monocyte differentiation into macrophages and cyclooxygenase-2 (Cox-2) expression, based upon the observation that high amounts of this enzyme, colocalizing mainly with macrophages, have been found in human atherosclerotic lesions. Moreover, the hypothesis that reactive oxygen species (ROS) could be important as mediators of Cox-2 expression during monocyte differentiation was verified. Although ROS are known as modulators of gene expression profile, their involvement in monocyte differentiation has not been explored previously. METHODS: Human adherent monocytes and the promonocytic cell line U937 were differentiated into macrophages by phorbol ester (PMA). Cox-2 was evaluated in terms of protein, mRNA and activity. Intracellular ROS formation was measured by the oxidant sensitive dye 2',7'-dichlorofluorescein diacetate. NADPH oxidase subunit p47(phox) was evaluated by Western blot analysis. RESULTS: Functionally active Cox-2 is expressed during PMA-induced monocyte transition into macrophages and ROS driven by the NADPH oxidase play a critical role in this event. CONCLUSION: Monocyte differentiation into macrophages, possibly triggered by unquenched ROS, may contribute to the increased inflammatory response within atheromata.

Oxidized phospholipids inhibit cyclooxygenase-2 in human macrophages via nuclear factor-kappaB/IkappaB- and ERK2-dependent mechanisms.

OBJECTIVE: Oxidized low-density lipoproteins (ox-LDL) or their components suppress macrophage inflammatory response by down-regulating cytokine synthesis, nitric oxide synthase and inducible cyclooxygenase (Cox-2). This event is crucial for the pathophysiological process leading to the formation of atherosclerotic plaque. Our present study focused on the mechanisms through which oxidized phospholipids inhibit LPS-induced Cox-2 expression in human macrophages. METHODS: Macrophages were incubated with a mixture of oxidized fragmented phospholipids (ox-PAPC), present in modified LDL, and then exposed to LPS. Cox-2 was evaluated in terms of protein levels, mRNA and activity. RESULTS: Ox-PAPC dose-dependently inhibited Cox-2 protein, mRNA and activity by preventing NF-kappaB binding to DNA. This effect was consequent to alterations of the degradation pattern of IkappaBalpha. Moreover, ox-PAPC markedly prevented extracellular signal-regulated kinase (ERK2) activation, leading to Cox-2 expression, whereas activation of the transcription factor peroxisome proliferator-activated receptors (PPARs) was not influenced. CONCLUSION: ox-PAPC down-regulates LPS-induced Cox-2 expression in human macrophages by targeting both NF-kappaB/IkappaB and ERK2 pathways. An altered inflammatory response by macrophages within atheromata may contribute to the progression of atherosclerosis.

Apocynin prevents cyclooxygenase 2 expression in human monocytes through NADPH oxidase and glutathione redox-dependent mechanisms.

In the present study we report the preventive effect of apocynin, an active constituent of the Himalayan herb Picrorhiza kurrooa, on cyclooxygenase-2 (Cox-2) synthesis and activity in human adherent monocytes exposed to serum treated zymosan (STZ) and phorbol myristate acetate (PMA). Apocynin markedly decreases the intracellular reduced/oxidized glutathione ratio (GSH/GSSG) and prevents nuclear factor-kappaB (NF-kappaB) activation in stimulated monocytes. Moreover, it reduces intracellular reactive oxygen species (ROS) generation, NADPH oxidase activity in monocyte homogenates and translocation of p47phox subunit in monocyte membranes. p47phox levels are also reduced in lysates of apocynin-treated monocytes. The inhibition of Cox-2 by apocynin is completely abrogated by GSH provision. Results from this study indicate that apocynin inhibits Cox-2 synthesis and activity induced in monocytes by an increased oxidative tone and provide an explanation for the protective effect exerted by this compound in numerous cell and animal models of inflammation. Attenuation of NADPH oxidase derived ROS coupled with GSH/GSSG reduction and suppression of NF-kappaB activation are highlighted as the molecular mechanisms responsible for Cox-2 inhibition.

Vascular thrombogenicity induced by progressive LDL oxidation: protection by antioxidants.

Oxidative modification of LDL, which dysregulates the homeostasis between blood and vascular cells, and alterations of endothelial function are considered among the early events in the pathogenesis of atherosclerosis. This study was designed to evaluate the impact of progressive LDL oxidation on the thrombotic response both in vitro and in vivo, and to address the potential effect of antioxidants. Tissue factor was induced by progressive LDL oxidation in HUVEC, and this event was in parallel to the appearance of the apoptotic phenotype. Both these phenomena were mediated by ERK1/2 activation and were prevented by LDL pre-enrichment with antioxidants. In contrast, antioxidants failed to affect tPA and PAI-1 secretion, which was increased by LDL, either native or oxidized. Tissue factor-pathway inhibitor was also increased upon HUVEC exposure to progressively oxidized LDL. LDL, in the presence of an oxidative agent, trigger a thrombogenic response in vivo, mostly TF-dependent, in an in situ model of platelet deposition. This effect was markedly attenuated when LDL were enriched with antioxidants. It can be concluded that vascular thrombogenicity is induced by progressive LDL oxidation and that alterations of the antioxidant/oxidant balance of the LDL particle in favor of the antioxidant tone are protective against the thrombotic response triggered by oxidative stress. The extrapolation of these data in a clinical setting, even if not easy, offers potential insights for the use of antioxidants in the prevention of thrombotic complications associated with atherothrombosis.

Coagulation and fibrinolytic markers in a two-month follow-up of coronary bypass surgery.

OBJECTIVES: The alterations of the coagulation-fibrinolytic profile immediately and up to few days after cardiac surgery have been widely documented. However, less information is available on whether these alterations persist for prolonged periods of time after the operation. In this study we have evaluated the coagulation-fibrinolytic profile of patients who underwent coronary artery surgery with cardiopulmonary bypass during a 2-month follow-up period. METHODS: Twenty-six patients (age range, 50-75 years) were studied. Blood samples were collected before the intervention and at different time points postoperatively up to 2 months after the operation. Measurement of selected coagulation-fibrinolytic variables was carried out in plasma from 16 patients. Evaluation of tissue factor activity determined as procoagulant activity was performed in peripheral blood mononuclear leukocytes obtained from 10 patients. RESULTS: Antigenic levels of clottable fibrinogen, prothrombin fragment F1.2, D-dimer, and thrombin-antithrombin complex were significantly increased during the first week after the intervention compared with preoperative values. Prothrombin fragment F1.2 levels returned to normal within 15 days, fibrinogen levels normalized within 30 days, and thrombin-antithrombin complex levels normalized at 45 days, whereas D-dimer values were still significantly higher 60 days postoperatively respective to baseline values. There was a trend toward an increased procoagulant activity from peripheral blood mononuclear leukocytes 4 days after the operation, whereas no changes of factor VII measured either as antigen or in its coagulant and activated forms were recorded throughout the study. CONCLUSIONS: A marked activation of the coagulation-fibrinolytic system occurs after cardiopulmonary bypass and lasts for at least 2 months thereafter. This finding suggests that these alterations might account for the increased thrombotic risk of these patients during the postoperative period.

Overcoming limitations of current antiplatelet drugs: a concerted effort for more profitable strategies of intervention.

Platelets play a central role in the pathophysiology of atherothrombosis, an inappropriate platelet activation leading to acute ischemic complications (acute myocardial infarction, ischemic stroke). In view of this, platelets are a major target for pharmacotherapy. Presently, the main classes of antiplatelet agents approved for the use in such complications are aspirin and thienopyridines. Although antiplatelet treatment with these two types of drugs, alone or in combination, leads to a significant reduction of non-fatal myocardial infarction (-32%), non-fatal stroke (-25%), and of cardiovascular death (-17%), a residual risk persists. Newer antiplatelet agents have addressed some, but not all, these limitations. Vis-à-vis their net clinical benefit, the higher potency of some of them is associated with a rise in bleeding complications. Moreover, newer thienopyridines do not show advantages over and above the older ones as to reduction of stroke. A concerted effort that takes into consideration clinical, genetic, and laboratory information is increasingly recognized as a major direction to be pursued in the area. The well-established road signs of clinical epidemiology will provide major information to define newer potentially useful targets for platelet pharmacology.

Effect of two doses of aspirin on thromboxane biosynthesis and platelet function in patients undergoing coronary surgery.

Early post-operative aspirin improves survival in patients undergoing coronary artery bypass graft (CABG). However, most patients do not benefit of aspirin after CABG, still remaining at risk of thrombotic events due to insufficient platelet inhibition, specifically via the thromboxane (TX) pathway. We evaluated the effect of two aspirin doses (100 or 325 mg daily, enteric coated formulations) on platelet function and TX biosynthesis in patients after CABG and assessed whether the incidence of residual platelet reactivity could be reduced by the higher dose. Fifty-six patients undergoing CABG were randomly assigned to 100 or 325 mg aspirin daily for five days in a prospective single-centre study. Treatment effect was assessed by measuring either platelet function (light-transmission aggregometry and point-of-care PFA-100(R)) or TX biosynthesis in collagen-stimulated platelets, serum, urine, and in lipopolysaccharide (LPS)-cultured whole blood (WB). An insufficient TX inhibition was observed with 100 mg aspirin but not with the higher dose. The different effect of the two doses was, however, highlighted by either TX (platelet- or serum-derived) or by PFA-100(R) but not by the other assays. In conclusion, early after CABG, the incidence of residual platelet activity was lower in patients who received 325 mg aspirin. Moreover, evidence was provided that different methods yield different results in the detection of aspirin resistance, rendering them not interchangeable.

Cyclooxygenase-2 mediates hydrogen peroxide-induced wound repair in human endothelial cells.

Cyclooxygenase-2 (Cox-2) metabolites produced by endothelial cells, particularly prostacyclin and prostaglandin E(2), profoundly affect vascular tone, regional blood flow, and angiogenesis. We have previously shown that reactive oxygen species induce Cox-2 expression in human endothelial cells (HUVEC), either on their own or as components of the signaling pathway triggered by TNFalpha, the prototypical inflammatory cytokine. Here we investigated the role of Cox-2 induced by hydrogen peroxide (H(2)O(2)), either exogenous or endogenously generated by TNFalpha, in the repair of a mechanically wounded HUVEC monolayer and probed the sources of H(2)O(2) that are involved in TNFalpha signaling and the pathways through which H(2)O(2) modulates Cox-2 expression. Results indicate that H(2)O(2)-induced Cox-2 activity participates in the repair of wounded monolayers. Both NADPH oxidase and the mitochondrial electron transport chain are involved in H(2)O(2) generation. Signaling triggered by H(2)O(2) for Cox-2 induction acts by increasing the protein tyrosine kinase phosphorylation that follows inhibition of protein phosphatase activity. The activation of p38 MAPK and its interaction in the inhibition of serine/threonine phosphatase activity are both critical steps in this event. We conclude that Cox-2 induced by H(2)O(2) plays an important role in promoting endothelial wound repair after injury, so that the cardioprotective effect of Cox-2 is due at least in part to its power of healing damaged endothelium.

Pharmacological characterization of 2NTX-99 [4-methoxy-N1-(4-trans-nitrooxycyclohexyl)-N3-(3-pyridinylmethyl)-1,3-benzenedicarboxamide], a potential antiatherothrombotic agent with antithromboxane and nitric oxide donor activity in platelet and vascular preparations.

Thromboxane (TX) A(2), prostacyclin (PGI(2)), and nitric oxide (NO) regulate platelet function and interaction with the vessel wall. Inhibition of TXA(2), implemented synthesis of PGI(2), and supply of exogenous NO may afford therapeutic benefit. 2NTX-99 [4-methoxy-N(1)-(4-trans-nitrooxycyclohexyl)-N(3)-(3-pyridinylmethyl)-1,3-benzenedicarboxamide], a new chemical entity related to picotamide, showed antithromboxane activity and NO donor properties. 2NTX-99 relaxed rabbit aortic rings precontracted with norepinephrine or U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy-prosta-5Z,13E-dien-1-oic acid; EC(50), 7.9 and 17.1 microM, respectively), an effect abolished by 10 microM 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ). 2NTX-99 inhibited arachidonic acid (AA)-induced washed platelet aggregation (EC(50), 9.8 microM) and TXB(2) formation (-71% at 10 microM), and its potency increased in the presence of aortic rings (EC(50), 1.4 microM). In whole rabbit aorta incubated with homologous platelets, AA caused contraction and TXA(2) formation, reduced by 2NTX-99 (10-40 microM): contraction, -28 and -47%, TXA(2) formation, -37 and -75.4%, respectively, with concomitant increase in PGI(2). 2NTX-99 (20-40 microM) inhibited U46619-induced aggregation in rabbit platelet-rich plasma (PRP) (-74 +/- 6.7 and -96 +/- 2.4%, respectively) and inhibited collagen-induced aggregation in human PRP (-48.2 +/- 10 and -79.2 +/- 6%), whereas ozagrel was ineffective. In human embryonic kidney 293 cells transfected with the TXA(2) receptor isophorm alpha receptor, 2NTX-99 did not compete with the ligand, [(3)H]SQ29,548 ([(3)H][1S-[1alpha,2beta(5Z),3beta,4alpha]]-7-[3-[[2-(phenylamino)-carbonyl]hydrazino]methyl]-7-oxabicyclo[2,2,1]-hept-2-yl]-5-heptanoic acid), or prevent inositol phosphate accumulation. After oral administration (50-250 mg/kg), 2NTX-99 inhibited TXA(2) production in rat clotting blood (-71 and -91%); at 250 mg/kg, an area under the curve, 0 to 16 h, of 149.5 h/microg/ml and a t(1/2) of 6 h were calculated, with a C(max) value of 31.8 +/- 8.2 microg/ml. An excellent correlation between plasma concentrations and TXA(2) inhibition occurs. 2NTX-99 controls platelet function and vessel wall interaction by multifactorial mechanisms and possesses therapeutic potential.