Human Blood Brain Barrier Model

The present invention relates to an immortalized human brain endothelial cell line that is useful as an in vitro model of the blood brain barrier. CLAIMS 1. An immortalized human brain endothelial cell line deposited at CNCM (Institut Pasteur) on October 7, 2004 under deposit number No. I-3308. 2. A cell from the cell line of claim 1. 3. A method of measuring blood brain barrier permeability of a test substance, which method comprises : - providing cells of claim 2 in one or more confluent monolayer(s) ; - incubating said test substance with the confluent monolayer(s) ; and - measuring the amount of said test substance permeated across the confluent monolayer(s). 4. The method of claim 3, wherein the amount of said test compound permeated across the confluent monolayer(s) is measured by passive diffusion or active transport. 5. A method for determining the toxicity of a test substance toward the blood brain barrier, which method comprises incubating said test substance with cells of claim 2 and assessing the viability of the cells. 6. Use of the cell line of claim 1 as an in vitro model of human blood brain barrier

Tobacco smoke cooperates with interleukin-1\u3b2 to alter \u3b2-catenin trafficking in vascular endothelium resulting in increased permeability and induction of cyclooxygenase-2 expression in vitro and in vivo

Cigarette smoking affects all phases of atherosclerosis from endothelial dysfunction to acute occlusive clinical events. We explored activation by exposure to tobacco smoke of two genes, \u3b2-catenin and COX-2, that play key roles in inflammation and vascular remodeling events. Using both in vivo and in vitro smoke exposure, we determined that tobacco smoke (TS) induced nuclear \u3b2-catenin accumulation and COX-2 expression and activity and moreover interacted with IL-1 \u3b2 to enhance these effects. Exposure of cardiac endothelial cells to tobacco smoke plus IL-1\u3b2 (TS/IL-1\u3b2) enhanced permeability of endothelial monolayers and disrupted membrane VE-cadherin/\u3b2-catenin complexes, decreased \u3b2-catenin phosphorylation, and increased phosphorylation of GSK-3\u3b2, Akt, and EGFR. Transfection of endothelial cells with \u3b2-catenin-directed small interferring RNA (siRNA) suppressed TS/IL-1\u3b2-mediated effects on COX-2 modulation. Inhibitors of EGFR and phosphatidylinositol-3- kinase also abolished both the TS/IL-1\u3b2-mediated modulation of the Akt/GSK-3\u3b2/\u3b2-catenin pathway and enhancement of COX-2 expression. Moreover, increased levels of Akt and GSK-3\u3b2 phosphorylation, nuclear \u3b2-catenin accumulation, COX-2 expression, and IL-1\u3b2 were observed in cardiovascular tissue of ApoE-/- mice exposed to cigarette smoke daily for 2 wk. Our results suggest a novel mechanism by which cigarette smoking can induce proinflammatory and proatherosclerotic effects in vascular tissu

Alzheimer’s disease and Down’s syndrome: Treating Two Paths to Dementia.

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Suppressing PTEN activity by tobacco smoke plus interleukin-1beta modulates dissociation of VE-cadherin/beta-catenin complexes in endothelium

Objectives. Tobacco smoke (TS) interacts with inflammatory cytokines to produce endothelial dysfunction. We hypothesized that interleukin-1\u3b2 (IL-1\u3b2) plus TS (TS/IL-1\u3b2) induces disassembly of endothelial junctional complexes of VE-cadherin/\u3b2-catenin by suppression of PTEN activity and investigated molecular mechanisms that modulate PTEN-deactivation in this situation. Methods and Results. TS/IL-1\u3b2 exposure, which disrupted adherens junctions and induced nuclear \u3b2-catenin accumulation, increased tyrosine phosphorylation (p-Tyr) of VE-cadherin and \u3b2-catenin, and reduced PTEN activity. Overexpression or silencing of PTEN modulated p-Tyr of both VE-cadherin and \u3b2-catenin, changed assembly of adherens junction complexes, and altered nuclear \u3b2-catenin accumulation. In addition, inhibiting ROS production stimulated by TS/IL-1\u3b2 decreased activation of Src, EGFR and p38MAPK, phosphorylation of PTEN, VE-cadherin and_-catenin, and abrogated the effect of TS/IL-1\u3b2 to disorganize adherens junctions, resulting in reduced endothelial permeability and decreased nuclear \u3b2-catenin accumulation. Finally, exposure of ApoE-/- mice to cigarette smoke\u2013induced phosphorylation of Src, EGFR, p-38MAPK, PTEN, and \u3b2-catenin, and disrupted VE-cadherin/\u3b2-catenin complexes in cardiovascular tissue. Conclusions. TS interaction with IL-1\u3b2 modulates PTEN activity though the ROS/Src/EGFR-p38MAPK pathway. PTEN deactivation is essential to increase VE-cadherin and \u3b2-catenin p-Tyr and to disassemble VE-cadherin/\u3b2-catenin membrane complexes, events that lead to accumulation of \u3b2-catenin within the nucleu

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

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