Docosahexaenoic acid improves the nitroso-redox balance and reduces VEGF-mediated angiogenic signaling in microvascular endothelial cells.
OBJECTIVE
Disturbances to the cellular production of nitric oxide (NO) and superoxide (O(2)(-)) can have deleterious effects on retinal vascular integrity and angiogenic signaling. Dietary agents that could modulate the production of these signaling molecules from their likely enzymatic sources, endothelial nitric oxide synthase (eNOS) and NADPH oxidase, would therefore have a major beneficial effect on retinal vascular disease. The effect of ω-3 polyunsaturated fatty acids (PUFAs) on angiogenic signaling and NO/superoxide production in retinal microvascular endothelial cells (RMECs) was investigated.


METHODS
Primary RMECs were treated with docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) for 48 hours. RMEC migration was determined by scratch-wound assay, proliferation by the incorporation of BrdU, and angiogenic sprouting using a three-dimensional model of in vitro angiogenesis. NO production was quantified by Griess assay, and phospho-eNOS accumulation and superoxide were measured using the fluorescent probe dihydroethidine. eNOS localization to caveolin-rich microdomains was determined by Western blot analysis after subfractionation on a linear sucrose gradient.


RESULTS
DHA treatment increased nitrite and decreased superoxide production, which correlated with the displacement of eNOS from caveolar subdomains and colocalization with the negative regulator caveolin-1. In addition, both ω-3 PUFAs demonstrated reduced responsiveness to VEGF-stimulated superoxide and nitrite release and significantly impaired endothelial wound healing, proliferation, and angiogenic sprout formation.


CONCLUSIONS
DHA improves NO bioavailability, decreases O(2)(-) production, and blunts VEGF-mediated angiogenic signaling. These findings suggest a role for ω-3 PUFAs, particularly DHA, in maintaining vascular integrity while reducing pathologic retinal neovascularization.
Matesanz
Nuria
N
Centre for Vision and Vascular Science, Queen's University Belfast, Northern Ireland, United Kingdom.
Park
Grace
G
McAllister
Hollie
H
Leahey
William
W
Devine
Adrian
A
McVeigh
Gary E
GE
Gardiner
Tom A
TA
McDonald
Denise M
DM
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002417:Cattle; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004281:Docosahexaenoic Acids / Q000494:pharmacology*; D015118:Eicosapentaenoic Acid / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005456:Fluorescent Dyes; D020287:In Situ Nick-End Labeling; D009389:Neovascularization, Pathologic / Q000473:pathology / Q000517:prevention & control*; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D015538:Nitrosation; D010084:Oxidation-Reduction; D012171:Retinal Vessels / Q000166:cytology; D015398:Signal Transduction / Q000187:drug effects*; D013481:Superoxides / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000633:toxicity*; D014945:Wound Healing / Q000187:drug effects
D005456:Fluorescent Dyes; D042461:Vascular Endothelial Growth Factor A; D013481:Superoxides; D004281:Docosahexaenoic Acids; D009569:Nitric Oxide; D015118:Eicosapentaenoic Acid; D052250:Nitric Oxide Synthase Type III