Inhibition of the myosin light chain kinase prevents hypoxia-induced blood-brain barrier disruption.
Increased mortality after stroke is associated with development of brain edema. The aim of the present study was to examine the contribution of endothelial myosin light chain (MLC) phosphorylation to hypoxia-induced blood-brain barrier (BBB) opening. Measurements of trans-endothelial electrical resistance (TEER) were performed to analyse BBB integrity in an in vitro co-culture model (bovine brain microvascular endothelial cells (BEC) and rat astrocytes). Brain fluid content was analysed in rats after stroke induction using a two-vein occlusion model. Dihydroethidium was used to monitor intracellular generation of reactive oxygen species (ROS) in BEC. MLC phosphorylation was detected using immunohistochemistry and immunoblot analysis. Hypoxia caused a decrease of TEER values by more than 40%, which was prevented by inhibition of the MLC-kinase (ML-7, 10 micromol/L). In addition, ML-7 significantly reduced the brain fluid content in vivo after stroke. The NAD(P)H-oxidase inhibitor apocynin (500 micromol/L) prevented the hypoxia-induced TEER decrease. Hypoxia-dependent ROS generation was completely abolished by apocynin. Furthermore, ML-7 and apocynin blocked hypoxia-dependent phosphorylation of MLC. Our data demonstrate that hypoxia causes a breakdown of the BBB in vitro and in vivo involving ROS and the contractile machinery.
Kuhlmann
Christoph R W
CR
Institute of Physiology and Pathophysiology, Johannes Gutenberg University of Mainz, Mainz, Germany. kuhlma@uni-mainz.de
Tamaki
Ryo
R
Gamerdinger
Martin
M
Lessmann
Volkmar
V
Behl
Christian
C
Kempski
Oliver S
OS
Luhmann
Heiko J
HJ
D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism; D001381:Azepines / Q000494:pharmacology*; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000201:enzymology* / Q000503:physiopathology; D001929:Brain Edema / Q000188:drug therapy / Q000378:metabolism / Q000503:physiopathology; D002417:Cattle; D002478:Cells, Cultured; D018920:Coculture Techniques; D017097:Electric Impedance; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D045604:Extracellular Fluid / Q000187:drug effects / Q000378:metabolism; D002534:Hypoxia, Brain / Q000188:drug therapy / Q000201:enzymology* / Q000503:physiopathology; D008297:Male; D018994:Myosin Light Chains / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology
D000098:Acetophenones; D001381:Azepines; D004791:Enzyme Inhibitors; D018994:Myosin Light Chains; D009281:Naphthalenes; D017382:Reactive Oxygen Species; C070571:ML 7; C056165:acetovanillone; D019255:NADPH Oxidases; D009219:Myosin-Light-Chain Kinase