Adiponectin Mediates Dietary Omega-3 Long-Chain Polyunsaturated Fatty Acid Protection Against Choroidal Neovascularization in Mice

Abstract

Purpose: Neovascular age-related macular degeneration (AMD) is a major cause of legal blindness in the elderly. Diets with omega3-long-chain-polyunsaturated-fatty-acid (ω3-LCPUFA) correlate with a decreased risk of AMD. Dietary ω3-LCPUFA versus ω6-LCPUFA inhibits mouse ocular neovascularization, but the underlying mechanism needs further exploration. The aim of this study was to investigate if adiponectin (APN) mediated ω3-LCPUFA suppression of neovessels in AMD.

Methods: The mouse laser-induced choroidal neovascularization (CNV) model was used to mimic some of the inflammatory aspect of AMD. CNV was compared between wild-type (WT) and Apn-/- mice fed either otherwise matched diets with 2% ω3 or 2% ω6-LCPUFAs. Vldlr-/- mice were used to mimic some of the metabolic aspects of AMD. Choroid assay ex vivo and human retinal microvascular endothelial cell (HRMEC) proliferation assay in vitro was used to investigate the APN pathway in angiogenesis. Western blot for p-AMPKα/AMPKα and qPCR for Apn, Mmps, and IL-10 were used to define mechanism.

Results: ω3-LCPUFA intake suppressed laser-induced CNV in WT mice; suppression was abolished with APN deficiency. ω3-LCPUFA, mediated by APN, decreased mouse Mmps expression. APN deficiency decreased AMPKα phosphorylation in vivo and exacerbated choroid-sprouting ex vivo. APN pathway activation inhibited HRMEC proliferation and decreased Mmps. In Vldlr-/- mice, ω3-LCPUFA increased retinal AdipoR1 and inhibited NV. ω3-LCPUFA decreased IL-10 but did not affect Mmps in Vldlr-/- retinas.

Conclusions: APN in part mediated ω3-LCPUFA inhibition of neovascularization in two mouse models of AMD. Modulating the APN pathway in conjunction with a ω3-LCPUFA-enriched-diet may augment the beneficial effects of ω3-LCPUFA in AMD patients.

Figure 1

Dietary ω-3 LCPUFA versus ω-6 LCPUFA suppressed laser-induced CNV. Eight-week-old C57BL/6J (WT) mice were fed ω-3 or ω-6 LCPUFA-enriched diets starting 1 week before and continuing until 1 week after laser-induced CNV (day 0). At day 7, choroidal neovessels were examined. (A) Representative neovessel lectin-stained (red) choroidal whole-mounts. The brackets in the top panel are shown at the bottom. (B) Significantly reduced lesion areas were found in ω-3 versus ω-6 LCPUFA-fed retinas. n = 12 to 14 mice per group. Unpaired t-test. Arrow (top): optic nerve head. Scale bar: 200 μm (top); 50 μm (bottom).

Figure 2

APN deficiency abolished dietary ω-3 LCPUFA protection against laser-induced CNV. Eight-week-old Apn^−/− and WT mice were fed ω-3 or ω-6 LCPUFA-enriched diets from 1 week before until 1 week after laser-induced CNV (day 0). At day 7, choroidal neovessels were examined. Representative lectin-stained (red) vessels in choroidal whole-mounts (A, C) and quantification of choroidal lesion area (B, D). The brackets in the top panel are shown at the bottom (A, C). Significantly larger lesions were observed in ω-3 LCPUFA-fed Apn^−/− versus WT mice but not in ω-6 LCPUFA-fed Apn^−/− versus WT mice (B, D). n = 9 to 14 mice per group (ω-3). n = 8 to 12 mice per group (ω-6). Unpaired t-test. Arrow (top): optic nerve head. Scale bar: 200 μm (top, panel A, C); 50 μm (bottom, panel A, C).

Figure 3

The APN pathway inhibited neovessel formation through the suppression of MMP expression in laser-induced CNV. (A) WT mice fed ω-3 versus ω-6 LCPUFA-enriched diets had increased Apn, and decreased Mmp2 and Mmp9, as well as unchanged AdipoR1 expression in choroidal-retinal explants. n = 3 mice per group. Unpaired t-test. (B) APN deficiency abolished ω-3 LCPUFA inhibition of choroidal-retinal Mmp2 and Mmp9 levels. n = 3 to 4 mice per group. Unpaired t-test. (C) APN deficiency decreased the levels of phosphorylated AMPKα in choroidal-retinal CNV explants. n = 6 mice per group. Unpaired t-test. (D) In the choroidal sprouting assay ex vivo, choroidal explants from Apn^−/− mice showed larger sprouting areas than WT mice. n = 4 to 6 explants per group. Unpaired t-test. Scale bar: 500 μm. (E) In HRMEC MTT assay in vitro, activation of the APN pathway with APN receptor agonist adipoRon inhibited endothelial cell proliferation. n = 10 replicates per group. Unpaired t-test. (F) AdipoRon treatment (25 μM) decreased Mmp2 and Mmp9 expression in HRMECs. Results were repeated in two independent experiments. Unpaired t-test.

Figure 4

Schematic of the APN pathway in mediating ω-3 LCPUFA inhibition on laser-induced CNV.

Figure 5

Dietary ω-3 LCPUFA decreased retinal neovascular lesions in Vldlr^−/− mice. Vldlr^−/− mice were fed either ω-3 or ω-6 LCPUFA-enriched diets from postnatal day (P) P1. (A) In Vldlr^−/− retinas, ω-3 LCPUFA induced gene expression of APN receptor adipoR1 but not Apn (n = 3 to 4 mice per group). Unpaired t-test. (B) Increased AdipoR1 mRNA levels in Vldlr^−/− versus Vldlr^+/+ retinas from postnatal day 3 to 17. Unpaired t-test. (C) At P16, retinal neovascularization was examined. 3D reconstruction demonstrated blood vessels invading from the OPL toward RPE; representative images of neovascular lesion (200× magnification). (D) Representative images of isolectin-stained vessels (red) in retinal whole mounts. (E) Dietary ω-3 versus ω-6 LCPUFA supplementation decreased the number and total lesion area of neovascular lesions (n = 7 to 10 mice per group). Unpaired t-test. (F) ω-3 versus ω-6 LCPUFA did not affect retinal Mmp2 and Mmp9 expression in Vldlr^−/− mice. Retinal IL-10 was increased in ω-3 LCPUFA-fed Vldlr^−/− mice. n = 3 to 4 mice per group. Unpaired t-test. (G) Change in IL-10 in Vldlr^−/− mice treated with either 50 mg/kg adipoRon or vehicle from P3 to 15. n = 3 to 4 retinas per group. (H) In the model of laser-induced CNV, there was no change in IL-10 expression in choroidal-retinal complex from WT mice fed ω-3 versus ω-6 LCPUFA-enriched diets. n = 3 mice per group. Unpaired t-test.