22. European Stroke Conference 4 Vascular biology 9:00 - 9:10 Inhibition of protein kinase C-beta reverses hyperglycaemia-induced in vitro cerebral barrier permeability through regulation of RhoA/Rho-kinase/MLC2 pathway K. Srivastava1, B. Shao2, U. Bayraktutan3 The University of Nottingham, Nottingham, UNITED KINGDOM1,The University of Not-tingham, Nottingham, UNITED KINGDOM2, The University of Nottingham, Nottingham, UNITED KINGDOM3 Background: Ischaemic stroke patients with hyperglycaemia (HG) develop higher volumes of brain oedema resulting from the disruption of blood-brain barrier (BBB). This study explored whether activation of protein kinase C-beta (PKC-beta) and RhoA/Rho-kinase/myosin-regula-tory light chain-2 (MLC2) pathway may in part account for HG-induced barrier hyperperme-ability. Methods: PKC-beta and RhoA activities were measured in human brain microvascular endothe-lial cells (HBMEC) by Peptag and Rhotekin-binding assays, respectively. The integrity of an in vitro model of human BBB comprising HBMEC and astrocytes was examined by assessments of transendothelial electrical resistance (TEER) and paracellular flux of Evan’s blue-labelled albumin (EBA). Protein expression and localisations were assessed by in-cell Western analyses and immunocytochemistry, respectively. Results: HG (25 mM D-glucose) significantly increased RhoA/Rho-kinase protein expressions, MLC2 phosphorylation and PKC-beta and RhoA activities in HBMEC while markedly reduc-ing the level of tight junction protein, occludin. HG-evoked in vitro barrier dysfunction, con-firmed by decreases in TEER and concomitant increases in flux of EBA, was accompanied by impairments of endothelial cytoskeleton and tight junctions. Suppression of RhoA and Rho-ki-nase activities by anti-RhoA antibody electroporation and Y-27632, respectively prevented morphological changes and restored plasma membrane localisation of occludin. Normalisation of glucose levels and silencing PKC-beta activity (via specific siRNA) neutralised the effects of HG on the expression, phosphorylation and activity of occludin and RhoA/Rho-kinase/MLC2 pathway components and consequently improved in vitro barrier integrity and function. Conclusions: These results suggest that HG-induced exacerbation of the BBB breakdown fol-lowing an ischaemic stroke is mediated in large part by activation of PKC-beta. 3 Vascular biology 8:50 - 9:00 The low-expression variant of fatty acid-binding protein 4 (FABP4) associates with as-ymptomatic carotid atherosclerosis J. Saksi1, P. Ijäs2, M.I. Mäyränpää3, K. Nuotio4, J. Tuimala5, M. Jauhiainen6, M. Kaste7, P.T. Kovanen8, P.J. Lindsberg9 Helsinki University Central Hospital, Department of Neurology, Helsinki, FINLAND1,Hel-sinki University Central Hospital, Department of Neurology, Helsinki, FINLAND2, Haartman Institute, Department of Pathology, Helsinki, FINLAND3, Helsinki University Central Hospital, Department of Neurology, Helsinki, FINLAND4, Finnish Red Cross Blood Service, Helsinki, FINLAND5, National Institute of Health and Welfare, Helsinki, FINLAND6, Helsinki Universi-ty Central Hospital, Department of Neurology, Helsinki, FINLAND7, Wihuri Research Institute, Helsinki, FINLAND8, Helsinki University Central Hospital, Department of Neurology, Helsin-ki, FINLAND9 Background: Fatty acid-binding protein 4 (FABP4) has been identified as a regulator of lipotox-ic endoplasmic reticulum (ER) stress in macrophages. Previously, we and others have shown FABP4 over-expression in stroke-susceptible carotid plaques (CP). A promoter polymorphism (T-87C, rs77878271) lowering FABP4 mRNA levels has been described. This led us to study this low-expression variant (C-var) in carotid atherosclerosis (CA). Methods: Rs77878271 was genotyped in patients with significant CA (HeCES, n=92) and in the Health 2000 Survey, an epidemiological cross-sectional health survey (n=7491) with carotid ultrasound data (n=1448). FABP4 expression was quantified from the CPs usinq qPCR and ELISA. Immunohistochem-istry was used to detect FABP4, ER stress markers, and to quantify apoptosis markers, activat-ed caspase 3 (ACA3) and TUNEL reactivity. Results: Asymptomatic CA patients had higher frequency of the C-var (0.14 vs 0.04, P=0.038). The C-var carriers showed 3.8-fold lower FABP4 mRNA levels (p=0.049) in the CPs, while high FABP4 expression was associated with lipid accumulation, plaque haemorrhages and ulcerations, apoptosis and phosphoactivated ER stress markers. The C-var associated with a 2.7-fold reduction in ACA3 immunoreactive cells (P=0.043) with a trend towards lower TUNEL reactivity. In the Health 2000 cohort, obese C-var carriers showed a reduction in carotid IMT (P=0.010) and less CPs (P=0.060). Homozy-gous C-var carriers showed a reduction in cardiovascular events (stroke, TIA, AMI and CHD), but this was not significant (3.6% vs. 13.1% in TT and 12.6% in TC carriers, P=0.167). Conclu-sions: Our data suggests that high FABP4 expression associates with carotid atherosclerosis and plaque vulnerability while reduced FABP4 expression may lead to a more stable plaque pheno-type with reduced cardiovascular risk by attenuating apoptosis and ER stress. These data sup-port the feasibility of FABP4 inhibition in the prevention of atherothrombotic complications. 28 © 2013 S. Karger AG, Basel Scientific Programme
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