Angiopoietin modulation of vascular endothelial growth factor: Effects on retinal endothelial cell permeability.
OBJECTIVE
Vascular permeability is important at many sites, but particularly so in diabetic retinopathy where macular oedema is the major cause of blindness. Angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) are important factors involved in neovascularization and vascular leakage, but there is little data on their interaction to promote increased vascular permeability.


METHODS
Porcine retinal endothelial cells (PREC) were seeded into permeable inserts and cultured in 24-well plates that permit measurement of permeability using fluorescent dextrans. Cell purity was assessed immunohistochemically. At confluency, PREC were treated with increasing concentrations of VEGF (20-100ng/ml) and Ang-2 (15-75ng/ml). The effect on tight junctions was assessed by visualization with an anti-ZO-1 antibody.


RESULTS
Immunohistochemistry showed high purity of isolated PREC. Permeability of untreated PREC monolayers was low. The increase in permeability in Ang-2 treated cells (25-30% compared with non-treated cells) was less than that for cells treated with VEGF only (20-100% compared with untreated cells). Highest permeability was seen with a combination of Ang-2 and VEGF (100-400% compared with untreated cells). Permeability increased with time after growth factor application. Preliminary ZO-1 immunohistochemistry appeared to demonstrate the presence of tight junctions between untreated PREC, and loss of tight junctions after treatment with VEGF and Ang-2.


CONCLUSIONS
VEGF alone is twice as potent in interrupting tight junctions in an endothelial cell monolayer as Ang-2. However, both growth factors acting together increase permeability three times as much as VEGF alone. Treatments designed to reduce vascular permeability in diabetic macular oedema should consider that crosstalk between growth factors including VEGF and the Ang-2/Tie-2 system can multiply their effects.
Peters
Swaantje
S
University College of London, Institute of Ophthalmology, Departments of Cell Biology and Pathology, 11-43 Bath Street, London, UK.
Cree
Ian A
IA
Alexander
Robert
R
Turowski
Patric
P
Ockrim
Zoe
Z
Patel
Jignesh
J
Boyd
Shelley R
SR
Joussen
Antonia M
AM
Ziemssen
Focke
F
Hykin
Philip G
PG
Moss
Stephen E
SE
D042702:Angiopoietin-2 / Q000819:agonists / Q000494:pharmacology*; D000818:Animals; D002199:Capillary Permeability / Q000187:drug effects*; D002478:Cells, Cultured; D003930:Diabetic Retinopathy / Q000378:metabolism* / Q000473:pathology; D004357:Drug Synergism; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology; D008269:Macular Edema / Q000378:metabolism / Q000473:pathology; D009389:Neovascularization, Pathologic / Q000378:metabolism / Q000473:pathology; D042787:Receptor, TIE-2 / Q000378:metabolism; D012160:Retina / Q000378:metabolism* / Q000473:pathology; D013552:Swine; D019108:Tight Junctions / Q000378:metabolism / Q000473:pathology; D042461:Vascular Endothelial Growth Factor A / Q000819:agonists / Q000494:pharmacology*
D042702:Angiopoietin-2; D042461:Vascular Endothelial Growth Factor A; D042787:Receptor, TIE-2