A short-crack growth model for corrosion-fatigue propagation in a mild steel exposed to artificial rainwater

One of the main problems for railway maintenance is the correct estimation of a safe service life for axles exposed to corrosion, since on one side there is no precise indications in existing standards while, on the other side, there is the very good experience of belgian railways of avoiding axle painting. In this paper we examine the applicability of some crack growth models for analysisng a data set of corrosion-fatigue crack growth data previously obtained onto the A1N railway axle steel under exposition to artificial rainwater. It will be shown that a fairly simple crack growth model could be easily estimated as a ’back-calculation’ from the S-N diagram. Application of the model to prediction of nucleation of ’technical crack’ under service load spectra will be also discussed

Inflammatory cytokines present in smokers\u2019 serum modulate cyclooxygenase-2 expression via NADPH-oxidase in endothelial cells

Background: Cigarette smoking modulates inflammation and endothelial dysfunction, events implicated in athero-thrombotic disease.1,2 Recently, endothelial dysfunction has been related to reactive oxygen species (ROS)3 and to prostanoids produced via cyclooxygenase-2 (COX-2)4. We hypothesized that inflammatory cytokines presents in smokers\u2019 serum induce ROS generation in endothelium by activation of NADPH oxidase with consequent COX-2 expression. Methods: Serum levels of interleukin-1beta (IL-1) and tumor necrosis factor alpha (TNF) were measured in 18 male volunteers (9 smokers and 9 non smokers). Human bone marrow endothelial cells (HBMEC) were exposed to serum from smokers or non-smokers, and ROS production (dichlorofluorescein fluorescence), NADPH oxidase activation (translocation of p47phox), COX-2 protein (Western blot) and mRNA (quantitative RT-PCR) were evaluated. Results: Serum from smokers compared with non smokers showed higher levels of IL-1 (P<0.05) and TNF (P<0.0008) and greater ability to induce ROS generation (P<0.002), translocation of p47phox from cytoplasm to plasma membrane (P<0.01), and increase COX-2 mRNA and protein (P<0.002) in HBMEC. Serum-induced ROS production and COX-2 mRNA by HBMEC correlated positively with IL-1 (r=0.723, P<0.002 and r=0.707, P<0.002) and TNF ( =0.781, P<0.0002 and r=0.772, P<0.0003). Moreover, there was a significant positive correlation between ROS generation and COX-2 mRNA (r=0.822, P<0.006). Simultaneous immunoneutralization of IL and TNF prevented ROS formation and COX-2 expression induced by smoker\u2019s serum. Inhibitors of NADPH oxidase and p47phox siRNA dramatically diminished smokers\u2019 serum-mediated endothelial ROS production and COX-2 expression. Conclusion: Our results suggest an essential role of inflammatory cytokines in the modulation of endothelial dysfunction induced by smoking. References 1. J. A. Ambrose et R. S. Barua. The pathophysiology of cigarette smoking and cardiovascular disease: an update. J. Am. Coll. Cardiol., 43, 1731-1737, 2004. 2. P.W. Wilson. Smoking, smoking cessation, and risk of cardiovascular disease. Curr. Treat. Options Cardiovasc. Med., 8, 276-281, 2006. 3. S.S. Barbieri, L. Ruggiero, E. Tremoli, B.B. Weksler. Suppressing PTEN activity by tobacco smoke plus interleukin-1beta modulates dissociation of VE-cadherin/beta-catenin complexes in endothelium. Arterioscler Thromb Vasc Biol., 28, 732-738, 2008 4. S. S. Barbieri et B. B Weksler. Tobacco smoke cooperates with interleukin-1 to alter \u3b2-catenin trafficking in vascular endothelium resulting in increased permeability and induction of ciclooxigenase-2 expression in vitro and in vivo. FASEB J., 21, 1-13, 200

Tobacco smoke regulates the expression and activity of microsomal prostaglandin E synthase-1 : role of prostacyclin and NADPH-oxidase

Tobacco smoke (TS) interacts with interleukin-1\u3b2 (IL-1\u3b2) to modulate generation of reactive oxygen species (ROS) and expression of cyclooxygenase-2. We explored molecular mechanisms by which TS/IL-1\u3b2 alters expression and activity of microsomal-prostaglandin E synthase-1 (mPGES-1) and of prostacyclin synthase (PGIS) in mouse cardiac endothelial cells. TS (EC(50) 3c5 puffs/L) interacting with IL-1\u3b2 (2 \u3bcg/L) up-regulates PGE(2) production and mPGES-1 expression, reaching a plateau at 4-6 h, but down-regulates prostacyclin (PGI(2)) release by increasing IL-1\u3b2-mediated PGIS tyrosine nitration. Inhibition of NADPH-oxidase, achieved pharmacologically and/or by silencing its catalytic subunit p47phox, or exogenous PGI(2) (carbaprostacyclin; IC(50) 3c5 \u3bcM) prevents production of both ROS and PGE(2), and negatively modulates mPGES-1 expression induced by TS/IL-1\u3b2. Moreover, inhibiting PGI(2), either using PGIS siRNA and/or CAY10441 (EC(50) 3c20 nM), a PGI(2) receptor antagonist, increases NADPH-oxidase activation, mPGES-1 synthesis, and PGE(2) production. Finally, lower PGI(2) levels associated with higher PGIS tyrosine nitration, p47phox translocation to the membrane (an index of activation of NADPH-oxidase), and mPGES-1 expression and activity were detected in cardiovascular tissues of ApoE(-/-) mice exposed to cigarette smoke compared to control mice. In conclusion, cigarette smoke in association with cytokines alters the balance between PGI(2)/PGE(2), reducing PGI(2) production and increasing synthesis and activity of mPGES-1 via NADPH-oxidase activation, predisposing to development of pathological conditions.-Barbieri, S. S., Amadio, P., Gianellini, S., Zacchi, E., Weksler, B. B., Tremoli, E. Tobacco smoke regulates the expression and activity of microsomal prostaglandin E synthase-1: role of prostacyclin and NADPH-oxidase

Cytokines present in smokers' serum interact with smoke components to enhance endothelial dysfunction

AIMS: Cigarette smoking engenders inflammation and endothelial dysfunction, processes implicated in atherothrombotic disease. We hypothesized that an interaction between inflammatory cytokines in smokers' blood and circulating components of cigarette smoke is necessary to induce reactive oxygen species (ROS) and cyclooxygenase-2 (COX-2) in endothelium. We then explored the molecular mechanisms involved in these effects. METHODS AND RESULTS: Serum from nine healthy active smokers (AS) compared with serum from nine non-smokers (NS) showed higher levels of interleukin-1beta (IL-1\u3b2) and tumour necrosis factor-alpha (TNF-\u3b1) and a greater ability to induce ROS production, p47phox translocation to the plasma membrane, and COX-2 mRNA and protein expression in endothelial cells (ECs). Similar results were obtained in vivo and in vitro after treatment with aqueous extracts of cigarette smoke plus IL-1\u3b2 and TNF-\u3b1(TS/IL-1\u3b2/TNF-\u3b1). In ECs increased ROS production and COX-2 mRNA induced by serum from AS correlated positively with their serum levels of IL-1\u3b2 and TNF-\u3b1. Moreover, a positive correlation was observed between ROS generation and COX-2 mRNA. Simultaneous immuno-neutralization of IL-1\u3b2 and TNF-\u3b1 prevented endothelial dysfunction induced by serum from AS. Inhibitors of NADPH oxidase and/or p47phox siRNA diminished ROS production and COX-2 expression as well as phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and Akt mediated either by AS serum or by TS/IL-1\u3b2/TNF-\u3b1. Finally, direct inhibition of p38MAPK and Akt activity also abolished COX-2 expression mediated by both types of stimuli. Our results suggest a crucial role played by interactions between inflammatory cytokines and tobacco smoke in the induction of endothelial dysfunction

Cigarette smoke-induced imbalance between PGE2/PGI2 modulates endothelial tissue factor : role of EP1 receptor and SIRT-1

Cigarette smoke exposure increases the incidence of atherothrombotic disease1,2, inducing expression of both cyclooxygenase-2 (COX-2)3, a key enzyme in the inflammatory response, and Tissue Factor (TF)4, initiator of the coagulation cascade. We assessed whether and how the major products of COX-2 activity (PGE2 and PGI2) modulate TF induced by cigarette smoke in endothelial cells (EC). We observed that an aqueous extract of cigarette smoke (TS) in association with inflammatory cytokine IL-1\uf062\uf020 altered the balance between PGE2/PGI2, increasing PGE2 and reducing PGI2 production, and induced TF (expression and activity) in EC in vitro. Inhibition of PGE synthase (PGES) activity by CAY10526, or by specific PGES siRNA, markedly diminished the amount of TF induced by TS/IL-1\uf062. In contrast, treatment with exogenous PGE2 increased TF. EC express three PGE2 receptors: EP1, EP2 and EP4. We showed that EP1 antagonists (AH6809 and SC19220) reduced TF induced by TS/IL-1\uf062 whereas an EP1 agonist (17-phenyl-trinor-PGE2,) increased TF. We excluded the involvement of other EP receptors because an EP4 antagonist (GW627368X) and EP2/EP4 agonists (misoprostol, butaprost) did not modify TF. The role of SIRT1, the NAD+-dependent protein deacetylase, in the regulation of TF was analyzed. Sirtinol, a SIRT1 deactivator, increased TF expression; in contrast, resveratrol, a SIRT1 activator, reduced TF induced by both TS/IL-1\uf062\uf020 and EP1 agonist. Furthermore, carbacyclin, a stable PGI2 receptor (IP) agonist, prevented TF and reduced PGE2 production induced by TS/IL-1\uf062\uf02e Conversely, both the IP antagonist, CAY10441, and specific PGI2 synthase siRNA increased both TF and PGE2. Finally, we showed that carbacyclin prevented TF expression induced by both PGE2 and Sirtinol. We conclude that cigarette smoke, by modulating the balance between PGE2/PGI2, increases expression and activity of TF by a pathway dependent on EP1/SIRT-1 (Fig. 1). Figure 1: The pathway induced by TS/Il-1\u3b2. References [1] J. A. Ambrose et R. S. Barua. The pathophysiology of cigarette smoking and cardiovascular disease: an update. J. Am. Coll. Cardiol., 43, 1731-1737, 2004. [2] P.W. Wilson. Smoking, smoking cessation, and risk of cardiovascular disease. Curr. Treat. Options Cardiovasc. Med., 8, 276-281, 2006. [3] S. S. Barbieri et B. B Weksler. Tobacco smoke cooperates with interleukin-1 to alter \u3b2-catenin trafficking in vascular endothelium resulting in increased permeability and induction of ciclooxigenase-2 expression in vitro and in vivo. FASEB J., 21, 1-13, 2007. [4] S. Matetzky, S. Tani, S. Kangavari, P. Dimayuga, J. Yano, H. Xu, K.Y. Chyu, M.C. Fishbein, P.K. Shah, B. Cercek. Smoking increases tissue factor expression in atherosclerotic plaques: implications for plaque thrombogenicity. Circulation, 102, 602-604,200

Prostacyclin reduction results in tissue factor (TF) upregulation and predisposes cyclooxygenase-2 knockout (COX-2KO) mice to thrombosis

Selective inhibitors of COX-2 increase the risk of myocardial infarction and athero-thrombotic events, but the mechanisms are not fully understood1,2. To assess the role of COX-2 in arterial thrombosis, a well-studied, chemically-induced model of arterial injury was applied to COX-2KO and data were compared with those obtained in wild-type (WT) mice. We found that thrombus formation in response to ferric chloride injury of the carotid arteries was significantly increased in COX-2KO than WT mice (Fig1). Figure 1: Kinetic of thrombus formation induced in response to ferric chloride injury of the carotid arteries in WT and COX-2KO mice. To understand the mechanisms responsible for the increase in thrombus formation, we first investigated the role of platelets in our experimental model. COX-2KO platelets showed enhanced response to collagen and ADP ex vivo in terms of aggregation together with increased ability to produce thromboxane compared with WT platelets. In this condition, COX-1 and thromboxane synthase protein levels were similar in both animal groups. However, using cross-transfusion experiments, we excluded that COX-2KO platelets were the responsible for the increased arterial thrombus formation in COX-2KO mice. Importantly, we found that the activity of tissue factor (TF) as an initiator of blood coagulation was elevated in concentrated microparticles derived from plasma as well as in leukocytes and in the carotid arteries of COX-2KO mice. Increased levels of TF mRNA correlated with reduced levels of prostacyclin synthase mRNA and decreased prostacyclin production in the arterial wall of COX-2KO mice. In addition, treatment with CAY10441, an antagonist of prostacyclin receptor (IP), increased TF activity in the carotid arteries of WT mice. Conversely, carbacyclin, a stable IP agonist, reduced TF activity in the carotid arteries of COX-2KO mice. These findings reveal for the first time that the propensity to thrombosis observed in association with COX-2 inhibition is consequent to the impairment in the generation of prostacyclin, which in turn results in increased TF expression and activity. References [1]D. Bishop-Bailey, J.A. Mitchell, T.D. Warner. COX-2 in Cardiovascular Disease. Arterioscler Thromb Vasc Biol, 26, 956-958, 2006. [2]S.D. Solomon, J.J.V. McMurray, M.A. Pfeffer, J.Wittes, R.Fowler, P.Finn, W.F.Anderson, A. Zauber, E. Hawk and Monica Bertagnolli. Cardiovascular Risk Associated with Celecoxib in a Clinical Trial for Colorectal Adenoma Prevention. N Engl J Med, 352, 1071-1080,200

Cyclooxygenase-2-derived prostacyclin regulates arterial thrombus formation by suppressing tissue factor in a sirtuin-1-dependent-manner

BACKGROUND: Selective inhibitors of cyclooxygenase (COX)-2 increase the risk of myocardial infarction We found that ferric chloride-induced arterial thrombus formation was significantly and thrombotic events, but the responsible mechanisms are not fully understood. METHODS AND RESULTS: greater in COX-2 knockout compared with wild-type mice. Cross-transfusion experiments excluded the likelihood that COX-2 knockout platelets, despite enhanced aggregation responses to collagen and thrombin, are responsible for increased arterial thrombus formation in COX-2 knockout mice. Importantly, we observed that COX-2 deletion decreased prostacyclin synthase and production and peroxisome proliferator-activated receptor- and sirtuin-1 (SIRT1) expression, with consequent increased upregulation of tissue factor (TF), the primary initiator of blood coagulation. Treatment of wild-type mice with a prostacyclin receptor antagonist or a peroxisome proliferator-activated receptor-\u3b4 antagonist, which predisposes to arterial thrombosis, decreased SIRT1 expression and increased TF activity. Conversely, exogenous prostacyclin or peroxisome proliferator-activated receptor-\u3b4 agonist completely reversed the thrombotic phenotype in COX-2 knockout mice, restoring normal SIRT1 levels and reducing TF activity. Furthermore, inhibition of SIRT1 increased TF expression and activity and promoted generation of occlusive thrombi in wild-type mice, whereas SIRT1 activation was sufficient to decrease abnormal TF activity and prothrombotic status in COX-2 knockout mice. CONCLUSIONS: Modulation of SIRT1 and hence TF by prostacyclin/peroxisome proliferator-activated receptor-\u3b4 pathways not only represents a new mechanism in controlling arterial thrombus formation but also might be a useful target for therapeutic intervention in the atherothrombotic complications associated with COX-2 inhibitors