VEGF increases endothelial permeability by separate signaling pathways involving ERK-1/2 and nitric oxide.
We tested the hypothesis that VEGF regulates endothelial hyperpermeability to macromolecules by activating the ERK-1/2 MAPK pathway. We also tested whether PKC and nitric oxide (NO) mediate VEGF-induced increases in permeability via the ERK-1/2 pathway. FITC-Dextran 70 flux across human umbilical vein endothelial cell monolayers served as an index of permeability, whereas Western blots assessed the phosphorylation of ERK-1/2. VEGF-induced hyperpermeability was inhibited by antisense DNA oligonucleotides directed against ERK-1/2 and by blockade of MEK and Raf-1 activities (20 microM PD-98059 and 5 microM GW-5074). These blocking agents also reduced ERK-1/2 phosphorylation. The PKC inhibitor bisindolylmaleimide I (10 microM) blocked both VEGF-induced ERK-1/2 activation and hyperpermeability. The NO synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (200 microM) and the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidiazoline-1-oxyl-3-oxide (100 microM) abolished VEGF-induced hyperpermeability but did not block ERK-1/2 phosphorylation. These observations demonstrate VEGF-induced hyperpermeability involves activation of PKC and NOS as well as Raf-1, MEK, and ERK-1/2. Furthermore, our data suggest that ERK-1/2 and NOS are elements of different signaling pathways in VEGF-induced hyperpermeability.
Breslin
Jerome W
JW
Program in Vascular Biology and Division of Vascular Surgery, Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine of New Jersey, Newark 07101-1709, USA.
Pappas
Peter J
PJ
Cerveira
Joaquim J
JJ
Hobson
Robert W
RW
Durán
Walter N
WN
D002199:Capillary Permeability / Q000187:drug effects*; D002478:Cells, Cultured; D016228:Endothelial Growth Factors / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D008222:Lymphokines / Q000494:pharmacology*; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D009569:Nitric Oxide / Q000494:pharmacology / Q000502:physiology*; D010766:Phosphorylation; D015398:Signal Transduction / Q000502:physiology; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D009569:Nitric Oxide; D020928:Mitogen-Activated Protein Kinases