Short communication: PPAR gamma mediates a direct antiangiogenic effect of omega 3-PUFAs in proliferative retinopathy

Abstract

Rationale: Omega3 long-chain polyunsaturated fatty acids (omega3-PUFAs) are powerful modulators of angiogenesis. However, little is known about the mechanisms governing omega3-PUFA-dependent attenuation of angiogenesis.

Objective: This study aims to identify a major mechanism by which omega3-PUFAs attenuate retinal neovascularization.

Methods and results: Administering omega3-PUFAs exclusively during the neovascular stage of the mouse model of oxygen-induced retinopathy induces a direct neovascularization reduction of more than 40% without altering vasoobliteration or the regrowth of normal vessels. Cotreatment with an inhibitor of peroxisome proliferator-activated receptor (PPAR)gamma almost completely abrogates this effect. Inhibition of PPARgamma also reverses the omega3-PUFA-induced reduction of retinal tumor necrosis factor-alpha, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, endothelial selectin, and angiopoietin 2 but not vascular endothelial growth factor.

Conclusions: These results identify a direct, PPARgamma-mediated effect of omega3-PUFAs on retinal neovascularization formation and retinal angiogenic activation that is independent of vascular endothelial growth factor.

Figures

Figure 1

ω3-PUFA intervention reduces neovascularization. (a) Whole retinal VEGF expression in normoxic retinas and retinas with OIR normalized to Cyclophilin A. (b) Quantification of retinal NV from mice with OIR at P15 and P17 and representative retinal flatmounts with NV in white (n≥8 per group) (c) Schematic of the mouse OIR model and ω3-PUFA intervention. (d) P17 Quantification of retinal NV and representative lectin-stained flatmounts from mice given either ω3- or ω6-PUFAs from P14–P17. (e) Quantification and representative flatmounts of retinal VO from the same mice at P17. Scale bars=500µm. n≥23 per group. *****p≤10−5. White areas signify NV in (d) and VO in (e).

Figure 2

PPARγ activation is required for ω3-mediated attenuation of neovascularization. (a) Quantification of retinal NV from mice with either ω3- or ω6-PUFA intervention and injection of either PPARγ-inhibitor GW9662 or vehicle from P14–P17. (b) Quantification of retinal VO from the same mice at P17. (c) Representative retinal flatmounts of P17 retinas for each experimental group (NV in white). Scale bars=500µm. n=10 per group. ****p≤0.001.

Figure 3

ω3-PUFA intervention reduces TNFα but not VEGF via PPARγ. (a) Retinal VEGF expression at P15 and P17 in mice with ω3- or ω6-PUFA intervention. n=6 per group (b) Retinal VEGF protein levels at P17 in mice with ω3- or ω6-PUFA intervention. n≥3 per group (c) Retinal flatmount from mice expressing GFP under the macrophage-specific Csf1r promoter (green) stained for endothelial cells with lectin (red) and TNFα (blue) at OIR P17. Scale bar=50µm. (d,e) Retinal and serum TNFα protein in mice with ω3-PUFA intervention and injection of either PPARγ-inhibitor GW9662 or vehicle from P14–P17. Vehicle-injected mice on ω6-PUFA served as controls. (f,g) TNFα and VEGF expression in RAW 264.7 macrophages in vitro with either docosahexaenoic acid (DHA), DHA+GW9662, or arachidonic acid (AA). n=3 independent wells per group **p≤0.03

Figure 4

ω3-PUFA intervention reduces endothelial cell activation via PPARγ. (a,b) Retinal P17 flatmount stained with isolectin (red) and (a) ICAM-1 (green) or (b) Ang-2 (green). Scale bar=50µm. (c) Schematic of retinal neovessels isolated using laser-capture microdissection. (d–g) Gene expression of ICAM-1, VCAM-1, E-Selectin and Ang-2 in P17 laser-captured retinal neovessels from mice with ω3-PUFA intervention and injection of either PPARγ-inhibitor GW9662 or vehicle from P14–P17. Vehicle-injected mice on ω6-PUFA served as controls. All data normalized to Cyclophilin A. (h) Western blot and quantification for retinal Ang-2 protein on mice from the same groups as in d–g. **p≤0.03, ***p≤0.01, ****p≤0.001