AMP kinase activation by Omega-3 polyunsaturated fatty acid protects the retina against ischemic insult: An in vitro and in vivo study

Abstract

PurposeTo investigate the possible beneficial effects of omega-3 polyunsaturated fatty acids (ω3-PUFAs) in ischemic retinal angiogenesis and whether AMP-activated protein kinase (AMPK) is involved. MethodsHuman retinal microvascular endothelial cells (hRMECs) were exposed to dimethyloxalylglycine (DMOG), a hypoxia-inducible factor hydroxylase inhibitor, in the presence or absence of docosahexaenoic acid (DHA) and small interfering RNA (siRNA) for AMPKα for 24 h. Ischemic factors, endothelial mesenchymal transition marker, endothelial barrier integrity, cell migration, and tube formation were evaluated. Neonatal AMPKα2−/− and control wild-type (WT) mice were submitted to an oxygen-induced retinopathy (OIR) protocol; their nursing mother mice were either fed ω3-PUFAs or not. In the end, ischemic markers and endothelial cell proliferation were evaluated in neonatal mouse retinal tissue through immunohistochemical or immunofluorescent assays among all studied groups. ResultsCells exposed to DMOG displayed increased expressions of hypoxic and endothelial mesenchymal transition (vimentin) markers and barrier disarrangement of Zonula Occludens-1 compared to the control, accompanied by increased cellular migration and tube formation (p < 0.05). AMPK activity was significantly decreased. Supplementation with DHA restored the mentioned alterations compared to DMOG (p<0.05). In siRNAAMPKα-treated cells, the beneficial effects observed with DHA were abolished. DHA upregulated G-protein receptor-120 (GPR120), which promptly increased intracellular levels of calcium (p ≤ 0.001), which consequently increased Calcium/calmodulin-dependent protein kinase kinase β expression (CaMKKβ) thus phosphorylating AMPKThr172. AMPKα2−/− and wild-type (WT) OIR mice exhibited similar retinal ischemic changes, and the oral supplementation with ω3-PUFA efficiently prevented the noticed ischemic alterations only in WT mice, suggesting that AMPKα2 is pivotal in the protective effects of ω3-PUFA. Conclusionsω3-PUFAs protect the retina from the effects of ischemic conditions, and this effect occurs via the GPR120-CaMKKβ-AMPK axis. A better understanding of this mechanism might improve the control of pathological angiogenesis in retinal ischemic diseases.