Antipermeability function of PEDF involves blockade of the MAP kinase/GSK/beta-catenin signaling pathway and uPAR expression.
OBJECTIVE
Pigment epithelium-derived factor (PEDF) is a potent inhibitor of vascular endothelial growth factor (VEGF)-induced endothelial permeability. The goal of this study was to understand the mechanism by which PEDF blocks VEGF-induced increases in vascular permeability.


METHODS
The paracellular permeability of bovine retinal endothelial (BRE) cells was measured by assaying transendothelial cell electrical resistance and tracer flux. Western blot analysis was used to show phosphorylation of VEGFR2, MAP kinases, and glycogen synthase kinase 3 (GSK3)-beta. Confocal imaging and Western blot analysis were used to determine subcellular distribution of beta-catenin. Real-time RT-PCR and Western blot analysis were used to quantify urokinase plasminogen activator receptor (uPAR) expression.


RESULTS
PEDF blocked VEGF-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 MAP kinase, the p38 substrate MAP kinase-activated protein kinase-2 (MAPKAPK-2), and GSK3-beta, but it had no effect on the phosphorylation of VEGFR2. In addition, the VEGF-induced transcriptional activation of beta-catenin and uPAR expression were blocked by PEDF and by inhibitors of p38 and MEK. Finally, the VEGF-induced increase in permeability was blocked by both PEDF and the same kinase inhibitors.


CONCLUSIONS
The data suggest that p38 MAP kinase and ERK act upstream of GSK/beta-catenin in VEGF-induced activation of the uPA/uPAR system and that PEDF-mediated inhibition of the VEGF-induced increase in vascular permeability involves blockade of this pathway. These findings are important for developing precise and potent therapies for treatment of diseases characterized by vascular barrier dysfunction.
Yang
Jinling
J
VA Medical Center, Augusta, Georgia, USA.
Duh
Elia J
EJ
Caldwell
Ruth B
RB
Behzadian
M Ali
MA
D000818:Animals; D015153:Blotting, Western; D002199:Capillary Permeability / Q000187:drug effects*; D002417:Cattle; D003911:Dextrans / Q000378:metabolism; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005136:Eye Proteins / Q000494:pharmacology*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009414:Nerve Growth Factors / Q000494:pharmacology*; D010766:Phosphorylation; D055293:Receptors, Urokinase Plasminogen Activator / Q000378:metabolism*; D012171:Retinal Vessels / Q000166:cytology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015843:Serpins / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology; D051176:beta Catenin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000099174:Pigment Epithelium-Derived Factor
D003911:Dextrans; D004791:Enzyme Inhibitors; D005136:Eye Proteins; D016650:Fluorescein-5-isothiocyanate; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D020928:Mitogen-Activated Protein Kinases; D009414:Nerve Growth Factors; D055293:Receptors, Urokinase Plasminogen Activator; D015843:Serpins; D042461:Vascular Endothelial Growth Factor A; D051176:beta Catenin; D000099174:Pigment Epithelium-Derived Factor; C015219:fluorescein isothiocyanate dextran