The role of RPE cell-associated VEGF₁₈₉ in choroidal endothelial cell transmigration across the RPE.
OBJECTIVE
To determine the role of vascular endothelial growth factor 189 (VEGF₁₈₉) in choroidal endothelial cell (CEC) migration across the retinal pigment epithelium (RPE) and to explore the molecular mechanisms involved.


METHODS
Using real-time PCR, the expression of VEGF splice variants VEGF₁₂₁, VEGF₁₆₅, and VEGF₁₈₉ was determined in human RPE from donor eyes, cultured human RPE in contact with CECs exposed to hydrogen peroxide (H₂O₂) or hypoxia, and RPE/choroid specimens from mice treated with laser to induce choroidal neovascularization (CNV). Activation of VEGF receptors (VEGFRs), phosphoinositol 3-kinase (PI-3K) or Rac1 was measured in CECs cocultured in contact with RPE exposed to peroxide or silenced for VEGF₁₈₉ expression. Migration of CECs across the RPE was determined using fluorescence microscopy.


RESULTS
VEGF₁₈₉ expression was increased in human RPE from aged compared with young donor eyes and from mouse RPE/choroids after laser to induce CNV. VEGF₁₈₉ was also upregulated in human RPE challenged with peroxide, hypoxia, or cultured in contact with CECs. CEC migration across RPE was greater after RPE exposure to peroxide to induce VEGF₁₈₉; VEGFR2 and Rac1 activities were also increased in these CECs. When CECs were cocultured with RPE silenced for VEGF₁₈₉, VEGFR2 and Rac1 activities in CECs were significantly reduced, as was CEC migration across the RPE. Inhibition of Rac1 activity significantly inhibited CEC transmigration without affecting PI-3K activity.


CONCLUSIONS
RPE-derived cell-associated VEGF₁₈₉ facilitates CEC transmigration by Rac1 activation independently of PI-3K signaling and may have importance in the development of neovascular AMD.
Wang
Haibo
H
Department of Ophthalmology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Geisen
Pete
P
Wittchen
Erika S
ES
King
Bradley
B
Burridge
Keith
K
D'Amore
Patricia A
PA
Hartnett
M Elizabeth
ME
D000328:Adult; D000368:Aged; D000375:Aging / Q000502:physiology; D000818:Animals; D002478:Cells, Cultured; D002829:Choroid / Q000098:blood supply*; D020256:Choroidal Neovascularization / Q000378:metabolism*; D018920:Coculture Techniques; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D020868:Gene Silencing / Q000502:physiology; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D057705:Transendothelial and Transepithelial Migration / Q000502:physiology*; D042461:Vascular Endothelial Growth Factor A / Q000502:physiology*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D055815:Young Adult; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D006861:Hydrogen Peroxide; D019869:Phosphatidylinositol 3-Kinases; D040301:Vascular Endothelial Growth Factor Receptor-2; D020830:rac1 GTP-Binding Protein
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