Extracellular signal-regulated protein kinase activation during reoxygenation is required to restore ischaemia-induced endothelial barrier failure.
During an ischaemic insult, oedema formation occurs as a consequence of increased vascular permeability. To study mechanisms leading to vascular barrier failure, endothelial cells were exposed to ischaemia (1% O(2) in serum- and glucose-free medium) for 5 h. In in vitro conditions, ischaemia increased paracellular permeability, disassembled actin stress fibres, displaced focal adhesion kinase (FAK) from focal adhesions and enhanced cytoskeletal association of occludin. Reoxygenation restored paracellular barrier function, actin organization and FAK distribution. The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) was rapidly activated after 30 min, strongly inhibited after 5 h of continuous ischaemia and reactivated 3 times more than control during reoxygenation. Inhibition of ERK activation during reoxygenation with U0126, an inhibitor of the ERK activator, MAPK/ERK kinase 1/2, prevented both barrier restoration and stress-fibre formation, but did not prevent recruitment of FAK to focal contacts. Under normoxic conditions, ERK inhibition led to barrier failure and disassembly of stress fibres only in the absence of serum. These results demonstrate that ERK activity is essential to rebuild a disrupted endothelial barrier after ischaemia and to maintain barrier function in cells exposed to non-ischaemic stress.
Institute of Biochemistry, Swiss Federal Institute of Technology, ETH Zentrum, CH-8092 Zurich, Switzerland.
Marco
M
Wachtel
Karl
K
Frei
Elisabeth
E
Ehler
Christian
C
Bauer
Max
M
Gassmann
Sergio M
SM
Gloor
D015153:Blotting, Western; D002463:Cell Membrane Permeability; D004730:Endothelium, Vascular / Q000201:enzymology / Q000378:metabolism* / Q000503:physiopathology; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D006801:Humans; D007511:Ischemia / Q000503:physiopathology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D011233:Precipitin Tests
D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010100:Oxygen