Cytochrome P450 2C8 ω3-long-chain polyunsaturated fatty acid metabolites increase mouse retinal pathologic neovascularization--brief report

Abstract

Objective: Regulation of angiogenesis is critical for many diseases. Specifically, pathological retinal neovascularization, a major cause of blindness, is suppressed with dietary ω3-long-chain polyunsaturated fatty acids (ω3LCPUFAs) through antiangiogenic metabolites of cyclooxygenase and lipoxygenase. Cytochrome P450 epoxygenases (CYP2C8) also metabolize LCPUFAs, producing bioactive epoxides, which are inactivated by soluble epoxide hydrolase (sEH) to transdihydrodiols. The effect of these enzymes and their metabolites on neovascularization is unknown.

Approach and results: The mouse model of oxygen-induced retinopathy was used to investigate retinal neovascularization. We found that CYP2C (localized in wild-type monocytes/macrophages) is upregulated in oxygen-induced retinopathy, whereas sEH is suppressed, resulting in an increased retinal epoxide:diol ratio. With a ω3LCPUFA-enriched diet, retinal neovascularization increases in Tie2-driven human-CYP2C8-overexpressing mice (Tie2-CYP2C8-Tg), associated with increased plasma 19,20-epoxydocosapentaenoic acid and retinal epoxide:diol ratio. 19,20-Epoxydocosapentaenoic acids and the epoxide:diol ratio are decreased with overexpression of sEH (Tie2-sEH-Tg). Overexpression of CYP2C8 or sEH in mice does not change normal retinal vascular development compared with their wild-type littermate controls. The proangiogenic role in retina of CYP2C8 with both ω3LCPUFA and ω6LCPUFA and antiangiogenic role of sEH in ω3LCPUFA metabolism were corroborated in aortic ring assays.

Conclusions: Our results suggest that CYP2C ω3LCPUFA metabolites promote retinal pathological angiogenesis. CYP2C8 is part of a novel lipid metabolic pathway influencing retinal neovascularization.

Keywords: angiogenesis factor; cytochrome P450 CYP2C8 (human); pathologic neovascularization.

Figure 1

Retinal expression of cytochrome P450 epoxygenases (CYP2C8) homologue, soluble epoxide hydrolase (sEH), and their products ratio in normoxia vs oxygen-induced retinopathy oxygen-induced retinopathy (OIR). A, Schematic diagram of CYP2C8 and sEH metabolism of arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA). B, Wild-type (WT) blood smear indicates CYP2C-positive leukocytes (arrows). Scale bar, 20 µm. C, mRNA level of CYP2C in blood and retina with or without perfusion. D, Three-dimensional (3D) reconstruction of confocal images of postnatal day (P) 17 WT normoxia and oxygen-induced retinopathy (OIR) retinal flat-mount stained with CYP2C (green), F4/80 (purple), isolectin (red), and 4',6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 100 µm. E, Layer-by-layer confocal image across a vein of normoxia retina. F, Colocalization of CYP2C and F4/80 (arrow) in OIR retinal flat-mount. G, Retinal cross-sectional staining with isolectin (red), CYP2C (green), and DAPI (blue) shows CYP2C is expression in neovascular tufts (arrowhead), as well as in the neurons of the ganglion cell layer (GCL), inner nuclear layers (INL), and outer nuclear layer (ONL). H, Retinal cross-sectional staining with isolectin (red), sEH (green), and DAPI (blue) shows sEH is expressed in neovascular tufts (arrow-head), as well as in neurons of GCL and INL. Scale bar, 10 µm. I, CYP2C and sEH mRNA expression in retina during OIR (n=6). J, CYP2C and sEH protein expression in normoxia (N) versus OIR (O) retina. K, The ratio of corresponding DHA and EPA epoxides to diols by liquid chromatography/mass spectrometry/mass spectrometry oxylipid analysis (n=4–6 per group; 2-way ANOVA with Bonferroni post test; *P<0.05). Three-dimensional reconstruction was made using Volocity 3D Image Analysis Software. All confocal images were taken by Leica SP2 confocal microscope with ×40 objective lens. Blood smear pictures was taken by Zeiss AxioObserver microscope under ×20 objective lens. DHET indicates dihydroxyeicosatrienoic acid; DiHDPA, dihydroxy-docosapentaenoic acid; EDP, epoxydocosapentaenoic acid; EET, epoxyeicosatrienoic acid; EEQ, epoxyeicosatetraenoic acid; and PUFA, polyunsaturated fatty acid.

Figure 2

With ω3-long-chain polyunsaturated fatty acid (ω3LCPUFA) feed, oxygen-induced retinopathy (OIR) neovascularization and corresponding epoxides level are modified in Tie2-CYP2C8-Tg and Tie2-sEH-Tg mice; the alternation of angiogenesis was also shown in aortic ring sprouting using Tie2-CYP2C8-Tg and Tie2-sEH-Tg treated with docosahexaenoic acid (DHA) and eicosapentaenoic acid (AA) or epoxide metabolites. A, Neovascular area of Tie2-CYP2C8-Tg mice exposed to OIR comparing with wild-type (WT) littermate control (n=11–13 per group). Scale bar, 500 µm. B, Neovascular area in OIR of Tie2-sEH-Tg comparing with WT (n=14–19 per group). C, Neovascular area in OIR of systemic soluble epoxide hydrolase (sEH) knockout (sEH−/−; n=8–15 per group). D and E, Reverse transcription polymerase chain reaction of vascular endothelial growth factor (VEGF)-A, VEGF-C, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in OIR Tie2-CYP2C8-Tg (D) and VEGF-A, VEGF-C in OIR Tie2-sEH-Tg (E) compared with WT (t test; *P<0.05 and **P<0.01). Retinal whole mount pictures were taken by Zeiss AxioObserver microscope under ×5 objective lens. F, Plasma levels of 19,20-epoxydocosapentaenoic acid (EDP) and 17,18-epoxyeicosatetraenoic acid (EEQ) in Tie2-CYP2C8-Tg mice (n=4–6 per group). G, Plasma levels of 19,20-EDP and 17,18-EEQ in Tie2-sEH-Tg mice (n=4–6 per group). H, Retinal 19,20-EDP:dihydroxy-docosapentaenoic acid (DiHDPA) and 17,18-EEQ:17,18-dihydroxy-eicosatetraenoic acid (DHEQ) ratio of Tie2-CYP2C8-Tg vs WT (n=4–6 per group). I, Retinal 19,20-EDP:DiHDPA and 17,18-EEQ:17,18-DHEQ ratio of Tie2-sEH-Tg vs WT (n=4–6 per group; t test; *P<0.05 and **P<0.01). J, Vehicle control, AA (30 µmol/L) or DHA (30 µmol/L) induced aortic ring sprouting of WT and Tie2-CYP2C8-Tg mice (n=3–7 per group). K, Aortic sprouting from Tie2-sEH-Tg and sEH−/− treated with 17,18-EDP and 19,20-EEQ (n=4–8 per group). Scale bars, 50 µm (t test; *P<0.05 and **P<0.01). Aortic ring pictures were taken by Zeiss AxioObserver microscope under ×10 objective lens. n.s. indicates not significant.