Volume 18, Issue 7, Pages 1606-1613 (February 2017) FGF21 Administration Suppresses Retinal and Choroidal Neovascularization in Mice  Zhongjie Fu, Yan Gong, Raffael Liegl, Zhongxiao Wang, Chi-Hsiu Liu, Steven S. Meng, Samuel B. Burnim, Nicholas J. Saba, Thomas W. Fredrick, Peyton C. Morss, Ann Hellstrom, Saswata Talukdar, Lois E.H. Smith  Cell Reports  Volume 18, Issue 7, Pages 1606-1613 (February 2017) DOI: 10.1016/j.celrep.2017.01.014 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 18, 1606-1613DOI: (10.1016/j.celrep.2017.01.014) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 FGF21 Treatment Decreased and FGF21 Deficiency Increased Hypoxia-Induced Retinal Neovascularization Quantification of neovascularization in P17 retinal whole mounts after oxygen-induced retinopathy. (A) Retinas were treated with native FGF21 (nFGF21), a long-acting FGF21 analog, PF-05231023, or vehicle control (n = 14–19 retinas per group; one-way ANOVA followed by Bonferroni’s multiple comparisons post test). n.s., not significant. (B) Retinas of Fgf21+/+ and Fgf21−/− mice (n = 12–13 retinas per group; unpaired t test). In whole mounts vessels were stained with isolectin (red) and neovascularization pseudo-colored white. Representative images are shown. Scale bar, 1 mm. Data are presented as mean ± SEM. Fold change compared with the control group was calculated. See also Figures S1 and S2. Cell Reports 2017 18, 1606-1613DOI: (10.1016/j.celrep.2017.01.014) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 FGF21 Suppressed Retinal Neovascularization via APN to Reduce Tnfα Quantification of neovascularization in P17 retinal whole mounts after oxygen-induced retinopathy. (A) WT P17 normal retina mRNA of FGF21 receptors Fgfr1, Fgfr2, Fgfr3, and Fgfr4 and co-receptor β-klotho (Klb) (n = 6–8 pooled retinas per group). (B) WT P17 hypoxic retinas and the effect of PF-05231023 on retinal Apn (n = 6 pooled retinas per group). PF-05231023 was administered from P12 to P16. (C) WT and Apn−/− mice (n = 17–20 retinas per group). (D) Apn−/− mice treated with PF-05231023 (n = 11–14 retinas per group). (E) Effect of PF-05231023 on Tnfα in WT or Apn−/− retinas (n = 6–8 pooled retinas per group). (F) Schematic of FGF21 modulation in neovascular retinas. In whole mounts, vessels were stained with isolectin (red), and neovascularization is pseudo-colored white. Representative images are shown. Scale bar, 1 mm. Data are presented as mean ± SEM (unpaired t test). n.s., not significant. Fold change was calculated. See also Figure S3. Cell Reports 2017 18, 1606-1613DOI: (10.1016/j.celrep.2017.01.014) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Exogenous FGF21 Decreased Retinal Neovascularization in Vldlr−/− Mice (A) 3D reconstruction of retinal neovessels extending from the outer plexiform layer (OPL) toward the retinal pigment epithelium (RPE). PF-05231023 (i.p. injected daily from P8 to P15) decreased retinal neovessel extension toward RPE seen with isolectin-stained vessels (red). (B) Representative images of isolectin-stained vessels in retinal whole mounts and neovessels (lesions) are highlighted in white (bottom) (with photoreceptor layer facing up). Scale bar, 1 mm. (C) The number and area (size) of vascular lesions were quantified (n = 11–18 retinas per group). (D) Effect of PF-0523102 on Apn and Tnfα in Vldlr−/− retinas (n = 4–6 pooled retinas per group) Data are presented as mean ± SEM (unpaired t test). Fold change was calculated. Cell Reports 2017 18, 1606-1613DOI: (10.1016/j.celrep.2017.01.014) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 FGF21 Administration Decreased Choroidal Neovascularization in Mice (A) Schematics of laser-induced choroidal neovascularization in mice. (B) Representative images of isolectin-stained choroidal neovessels. Area of lesions was quantified (n = 6–8 mice per group). Scale bars represent 200 μm (top) and 50 μm (bottom). (C) Effect of PF-05231023 on Apn and Tnfα in the neovascular choroid-retina complex (n = 4–6 pooled retinas per group). (D) Effect of PF-05231023 on Vegfa in the three mouse models of neovascular eye diseases (n = 6–8 pooled retinas per group). Data are presented as mean ± SEM (unpaired t test). n.s., not significant. Fold change was calculated. Cell Reports 2017 18, 1606-1613DOI: (10.1016/j.celrep.2017.01.014) Copyright © 2017 The Author(s) Terms and Conditions