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N. Matesanz, G. Park, H. McAllister, B. Leahey, A. Devine, G. McVeigh, T. A. Gardiner, D. M. McDonald; Omega-3 Fatty Acids Modulate Angiogenesis Through the Regulation of Nitric Oxide and Superoxide Anion Production in Retinal Microvascular Endothelial Cells (RMEC). Invest. Ophthalmol. Vis. Sci. 2009;50(13):2957.
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Previously, we have shown that the omega-3 polyunsaturated fatty acids (ω-3 PUFA) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) significantly decrease VEGF mediated angiogenic processes in isolated RMEC. The aim of this work was to investigate the mechanisms responsible for these effects, with a particular focus on the downstream mediators of VEGF function, nitric oxide (NO) and superoxide anion, produced from endothelial nitric oxide synthase (eNOS) and NADPH oxidase respectively. The subcellular targeting of eNOS to caveolae subdomains of the plasma membrane is important for the coupling of VEGF receptor activation to NO signaling events; thus, we also investigated the effect of DHA and EPA supplementation on eNOS caveolar localisation.
Primary cultures of RMEC were treated with 50 µM of EPA or DHA for 48 h and incorporation into cellular membranes verified by gas-liquid chromatography. NO production was quantified as nitrite accumulation by Griess assay and protein levels determined by western blotting. eNOS localisation to caveolae-rich microdomains of the plasma membrane was also determined by sub-fractionation on a linear sucrose gradient. Superoxide release was detected using the fluorescent probe dihydroethidine (DHE).
While EPA did not modify NO production with respect to untreated cells, preloading with DHA increased basal NO by 2.99±0.33 fold (p<0.001); eNOS protein content was unaffected and nNOS and iNOS expression was undetectable. The changes in NO production correlated with a displacement of eNOS from caveolae sub-compartments of the plasma membrane. When basal superoxide anion release was analysed, a decrease in EPA- and DHA-treated cells was observed (22.06% and 43.10%, respectively (p<0.001). In addition, VEGF- (20 ng/ml, 18 h) stimulated superoxide production (p<0.01), was also inhibited by superoxide dismutase (200 U/ml), the NADPH oxidase-inhibitor, apocynin (1 µM) and EPA or DHA treatment (p<0.001).
Here, we demonstrate that, ω-3 PUFA modulate the production of NO and superoxide by eNOS and NADPH oxidase, in part, by displacing eNOS from caveolae microdomains where VEGF receptor resides. Together, these findings suggest a mechanism by which these dietary fatty acids inhibit angiogenic processes in retinal endothelial cells.
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