The role of vascular endothelial growth factor-induced activation of NADPH oxidase in choroidal endothelial cells and choroidal neovascularization.
Rac1, a subunit of NADPH oxidase, plays an important role in directed endothelial cell motility. We reported previously that Rac1 activation was necessary for choroidal endothelial cell migration across the retinal pigment epithelium, a critical step in the development of vision-threatening neovascular age-related macular degeneration. Here we explored the roles of Rac1 and NADPH oxidase activation in response to vascular endothelial growth factor treatment in vitro and in a model of laser-induced choroidal neovascularization. We found that vascular endothelial growth factor induced the activation of Rac1 and of NADPH oxidase in cultured human choroidal endothelial cells. Further, vascular endothelial growth factor led to heightened generation of reactive oxygen species from cultured human choroidal endothelial cells, which was prevented by the NADPH oxidase inhibitors, apocynin and diphenyleneiodonium, or the antioxidant, N-acetyl-L-cysteine. In a model of laser-induced injury, inhibition of NADPH oxidase with apocynin significantly reduced reactive oxygen species levels as measured by dihydroethidium fluorescence and the volume of laser-induced choroidal neovascularization. Mice lacking functional p47phox, a subunit of NADPH oxidase, had reduced dihydroethidium fluorescence and choroidal neovascularization compared with wild-type controls. Taken together, these results indicate that vascular endothelial growth factor activates Rac1 upstream from NADPH oxidase in human choroidal endothelial cells and increases generation of reactive oxygen species, contributing to choroidal neovascularization. These steps may contributed to the pathology of neovascular age-related macular degeneration.
Monaghan-Benson
Elizabeth
E
Department of Cell and Developmental Biology, University of North Carolina, Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina, USA.
Hartmann
John
J
Vendrov
Aleksandr E
AE
Budd
Steve
S
Byfield
Grace
G
Parker
Augustus
A
Ahmad
Faisal
F
Huang
Wei
W
Runge
Marschall
M
Burridge
Keith
K
Madamanchi
Nageswara
N
Hartnett
M Elizabeth
ME
D000111:Acetylcysteine / Q000494:pharmacology; D000328:Adult; D000818:Animals; D015153:Blotting, Western; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D002829:Choroid / Q000098:blood supply* / Q000378:metabolism; D020256:Choroidal Neovascularization / Q000188:drug therapy / Q000209:etiology / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D006801:Humans; D007124:Immunoenzyme Techniques; D007834:Lasers / Q000009:adverse effects; D008268:Macular Degeneration / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology; D010750:Phosphoproteins / Q000502:physiology; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D009861:Onium Compounds; D010750:Phosphoproteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C079789:neutrophil cytosol factor 67K; C007517:diphenyleneiodonium; D019255:NADPH Oxidases; D020830:rac1 GTP-Binding Protein; D000111:Acetylcysteine
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