Increased dietary intake of omega-3-polyunsaturated fatty acids reduces pathological retinal angiogenesis

Abstract

Many sight-threatening diseases have two critical phases, vessel loss followed by hypoxia-driven destructive neovascularization. These diseases include retinopathy of prematurity and diabetic retinopathy, leading causes of blindness in childhood and middle age affecting over 4 million people in the United States. We studied the influence of omega-3- and omega-6-polyunsaturated fatty acids (PUFAs) on vascular loss, vascular regrowth after injury, and hypoxia-induced pathological neovascularization in a mouse model of oxygen-induced retinopathy. We show that increasing omega-3-PUFA tissue levels by dietary or genetic means decreased the avascular area of the retina by increasing vessel regrowth after injury, thereby reducing the hypoxic stimulus for neovascularization. The bioactive omega-3-PUFA-derived mediators neuroprotectinD1, resolvinD1 and resolvinE1 also potently protected against neovascularization. The protective effect of omega-3-PUFAs and their bioactive metabolites was mediated, in part, through suppression of tumor necrosis factor-alpha. This inflammatory cytokine was found in a subset of microglia that was closely associated with retinal vessels. These findings indicate that increasing the sources of omega-3-PUFA or their bioactive products reduces pathological angiogenesis. Western diets are often deficient in omega-3-PUFA, and premature infants lack the important transfer from the mother to the infant of omega-3-PUFA that normally occurs in the third trimester of pregnancy. Supplementing omega-3-PUFA intake may be of benefit in preventing retinopathy.

Figure 1

Elevated levels of ω-3-PUFAs result in decreased vaso-obliteration and retinal neovascularization in mice. Retinas from C57BL/6 mice fed an ω-3- or ω-6-PUFA enhanced diet were examined by whole-mount microscopy after oxygen-induced retinopathy. (a) P17 retinal vasculature stained with isolectin B4-FITC showing vaso-obliteration and neovascularization in the ω-6- and ω-3-PUFA fed mice (ω-6, n = 14, and ω-3, n = 27) (scale bar, 1 mm). (b) Vaso-obliteration and (c) neovascularization in the ω-3- and ω-6-PUFA-fed mice at P17. (d) Vaso-obliteration and neovascularization in wild-type (WT) versus Fat-1 retinas at P17 (scale bar, 1 mm). (e) Vaso-obliteration and (f) neovascularization in the fat-1-expressing mice compared with that in controls (wild-type, n = 20; Fat-1, n = 16). (g,h) Percentage vascularized area in retinal whole-mounts analyzed at P12, P13, P15 and P17 after exposure to 75% oxygen from P7–P12 from mice on a ω-6-PUFA or a ω-3-PUFA diet (g) or in Fat-1 mice or their wild-type controls (h), n = 8–20 per group. **P ≤ 0.001, ***P ≤ 0.0001; NS, not significant.

Figure 2

ResolvinD1, resolvinE1 and neuroprotectinD1, derived from ω-3-PUFAs, protect against retinopathy with reductions in vaso-obliteration and neovascularization. (a) Biosynthetic pathway of ω-3-PUFA metabolite formation from EPA and DHA. ω-22-Hydroxy-PD1 is a biosynthetic marker of NPD1. RvE2 is a biosynthetic marker of the E series EPA resolvins. (b,c) Liquid chromatography MS-MS spectrum of RvE2 (b) and of ω-22-hydroxy-PD1 (c) from retinal extracts of ω-3-PUFA-fed mice. (d) Vaso-obliteration at P17 in RvD1, RvE1 or NPD1-treated mice compared with vehicle-treated control. (e) Neovascularization at P17 in mice injected i.p. from P5–P8 with RvD1 (n = 14), RvE1 (n = 10) or NPD1 (n = 14) compared with that in saline-treated mice (n = 14). (f) Vaso-obliteration at P8 with RvD1 (n = 7), RvE1 (n = 9), NPD1 (n = 7) or saline (n = 6) treatment. (g) Left column, retinal whole-mounts from mice with oxygen-induced retinopathy at P14 on an ω-6-PUFA diet stained for endothelial cells (red), Csf1r (green) and ChemR23 (magenta). Central panel, merged image (scale bar, 50 μm); white indicates the colocalization of all three stains. Right column, four images of one Csf1r⁺ cell at indicated focal planes (z = 1.1 μm, 2.1 μm, 3.0 μm and 4.0 μm). The red lines on the cross-section (far right column) indicate the relative z depth that corresponds to the section shown directly to the left. ^#P ≤ 0.05; **P ≤ 0.0001.

Figure 3

Dietary PUFA regulation of TNF-α in retinopathy. (a) Left panel, mean total retinal Tnf mRNA expression in ω-6-PUFA and ω-3-PUFA-fed mice (n = 6) normalized to that of cyclophilin. Right panel, retinal TNF-α protein levels, normalized to total protein, at P14 in ω-6-PUFA and ω-3-PUFA-fed mice (n = 4). (b,c) Inhibition of ω-6-PUFA-induced retinopathy with suppression of TNF-α using etanercept. (b) Vaso-obliteration in etanercept treatment versus saline (i.p.) in ω-6-PUFA-fed (n = 8) and ω-3-PUFA-fed mice (n = 10) at P17. (c) Neovascularization in etanercept-treated ω-6-PUFA-fed mice versus saline-treated controls (n = 8) and in ω-3-PUFA-fed mice (n = 10) at P17. (d) Vaso-obliteration at P17 in Tnf^+/+ and Tnf^−/− mice on either an ω-3-PUFA diet (n = 15 and n = 11, respectively) or an ω-6-PUFA diet (n = 22 and n = 10, respectively). (e) Neovascularization at P17 in Tnf^+/+ and Tnf^−/− mice on either an ω-3-PUFA diet (n = 15 and n = 11, respectively) or an ω-6-PUFA diet (n = 22 and n = 10, respectively). (f) Retinal whole-mount from a P17 mouse on an ω-6-PUFA diet with oxygen-induced retinopathy stained (left column) for endothelial cells (red), Csf1r (green) and Tnf-α (magenta). Central panel, merged image of the three stains (scale bar, 50 μm). Right column, one Csf1r⁺ cell at focal planes z = 1.1 μm, 1.9 μm, 2.8 μm and 3.6 μm. Red lines (far right column) indicate relative z depth that corresponds to the section immediately to the left. (g) NF-κB (luciferase) activity (relative luminescence units) in P14 oxygen-treated retinas from NF-κB-Luc reporter mice on ω-6-PUFA (n = 10) or ω-3-PUFA (n = 11) diets. (h) Left column, retina with oxygen-induced retinopathy from a P14 mouse on an ω-6-PUFA diet stained for endothelial cells (red), CD11b (green) and luciferase (magenta). Right panel, merged image. Scale bar, 20 μm; ^#P ≤ 0.05, **P ≤ 0.001, ***P ≤ 0.0001.