22. European Stroke Conference Vascular Biology (PO 486 - 490) 486 Vascular biology TNF-alpha-induced cerebral barrier disruption during ischaemic injury is mediated by pro-oxidant enzyme NADPH oxidase Z. ABDULLAH1, U. BAYRAKTUTAN2 UNIVERSITY OF NOTTINGHAM, NOTTINGHAM, UNITED KINGDOM1, UNIVERSITY OF NOTTINGHAM, NOTTINGHAM, UNITED KINGDOM2 Background: Ischaemic injury is associated with excessive generations of TNF-alpha and oxidative stress and the disruption of blood-brain barrier (BBB). This study investigates whether pro-oxidant enzyme NADPH oxidase may modulate TNF-alpha-evoked BBB disruption. Methods: The levels of TNF-alpha were measured in human brain microvascular endothelial cells (HBMEC) after oxygen-glucose deprivation alone or followed by reperfusion (OGD±R) using an ELISA-based kit. Total superoxide anion (O2-) levels and NADPH oxidase activity were measured by cytochrome C reduction and lucigenin-enhanced chemiluminescence assays. The changes in pro-tein expressions and matrix metalloproteinase (MMP) activities were studied by Western analyses and gelatin zymography, respectively. The BBB integrity was assessed by measurements of transen-dothelial electrical resistance (TEER) and sodium fluorescein (NaF) flux across an in vitro model of human BBB composed of HBMEC and human astrocytes. Results: OGD±R produced marked increases in HBMEC TNF-alpha levels which in turn signifi-cantly enhanced total O2- levels and NADPH oxidase activity. TNF-alpha also increased MMP-2 and MMP-9 activities while concurrently reducing the expressions of antioxidant enzymes, Cu/ Zn-superoxide dismutase and catalase and the tight junction proteins, claudin-5 and occludin. Expo-sure of co-cultures to TNF-alpha led to dramatic disruptions in the BBB integrity as evidenced by reductions in TEER and concomitant increases in NaF flux. Inhibition of NADPH oxidase activity by one of the two structurally different inhibitors, namely apocynin or diphenyleneiodonium chlo-ride prevented all the aforementioned changes in protein expressions, enzyme activity and barrier integrity induced by TNF-alpha. Conclusion: During ischaemic cerebral injury, the activation of NADPH oxidase plays a pivotal role in determining the extent of TNF-alpha-evoked BBB damage through concomitant regulations of O2- bioavailability and MMP activity. 546 © 2013 S. Karger AG, Basel Scientific Programme 487 Vascular biology Lipoprotein associated phospholipase A2 (Lp-PLA2) and long-term prognosis after TIA and stroke H.C. Segal1, A.I. Burgess2, D.L. Poole3, N. Taj4, T.J. James5, Z. Mehta6, P.M. Rothwell7 Stroke Prevention Research Unit, Nuffield Department of Clinical Neurology, University of Oxford, Oxford, UNITED KINGDOM1, Stroke Prevention Research Unit, Nuffield Department of Clinical Neurology, University of Oxford, Oxford, UNITED KINGDOM2, Stroke Prevention Re-search Unit, Nuffield Department of Clinical Neurology, University of Oxford, Oxford, UNITED KINGDOM3, Clinical Biochemistry, Oxford University Hospitals NHS Trust, Oxford, UNITED KINGDOM4, Clinical Biochemistry, Oxford University Hospitals NHS Trust, Oxford, UNITED KINGDOM5, Stroke Prevention Research Unit, Nuffield Department of Clinical Neurology, Univer-sity of Oxford, Oxford, UNITED KINGDOM6, Stroke Prevention Research Unit, Nuffield Depart-ment of Clinical Neurology, University of Oxford, Oxford, UNITED KINGDOM7 BACKGROUND: Lipoprotein associated phospholipase A2 (Lp-PLA2), a serine lipase produced in macrophages and expressed in atherosclerotic lesions, participates in the oxidative modification of LDL. The released oxidised free fatty acids are potent pro-inflammatory products, potentially linking Lp-PLA₂ to cardiovascular disease and stroke. We determined the value of Lp-PLA2 in the prediction of recurrent vascular events and death after TIA or stroke. METHODS: We studied consecutive patients with TIA or stroke in a population-based study (Oxford Vascular Study) and community controls. Lp-PLA₂ was measured in serum samples using the PLAC test enzyme linked immunoassay for mass, and an automated colorimetric assay for activity (diaDexus Inc., San Fran-cisco, USA) the latter measured on the ADVIA 2400 (Siemens, Frimley Park, UK). In cases, Lp- PLA2 levels were related (OR per decile) to the 5-year risk of recurrent vascular events (ischaemic stroke and myocardial infarction) and risk of all-cause death after adjustment for age and sex. RE-SULTS: Among 1048 patients with TIA or stroke and 452 controls, Lp-PLA2 activity and mass were unrelated to age, but activity was lower (p=0.0001) in women than in men. Both Lp-PLA2 mean (SD) activity and mass were higher in patients than in controls (activity 184.8(49) vs 164.7(39) nmol/mL/min, p=0.0001; mass 294(79) vs 253(64)ng/mL), but there were no consistent differences between aetiological subtype (TOAST classification). Increased Lp-PLA2 mass tended to be associ-ated with risk of recurrent stroke (OR/decile=1.055, 0.999-1.114, p=0.054). Activity was unrelated to risk of recurrent stroke (1.008, 0.953-1.065, p=0.79), but did predict risk of death (1.057, 1.021- 1.095, p=0.0016). Neither activity nor mass predicted risk of MI. CONCLUSIONS: Lp-PLA2 as-says were not consistent predictors of recurrent vascular events after TIA or stroke. Further research is required to explain the link between Lp-PLA2 activity and all-cause mortality.
Karger_ESC London_2013
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