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. Author manuscript; available in PMC: 2017 Feb 27.
Published in final edited form as: Cell Rep. 2017 Feb 14;18(7):1606–1613. doi: 10.1016/j.celrep.2017.01.014

FGF21 administration suppresses retinal and choroidal neovascularization in mice

Zhongjie Fu 1, Yan Gong 1, Raffael Liegl 1, Zhongxiao Wang 1, Chi-Hsiu Liu 1, Steven S Meng 1, Samuel B Burnim 1, Nicholas J Saba 1, Thomas W Fredrick 1, Peyton C Morss 1, Ann Hellstrom 2, Saswata Talukdar 3,#, Lois EH Smith 1,#
PMCID: PMC5328201  NIHMSID: NIHMS843807  PMID: 28199833

Summary

Pathological neovascularization, a leading cause of blindness, is seen in retinopathy of prematurity, diabetic retinopathy and age related macular degeneration. Using a mouse model of hypoxia-driven retinal neovascularization we find that fibroblast growth factor 21 (FGF21) administration suppresses, and FGF21 deficiency worsens, retinal neovessel growth. The protective effect of FGF21 against neovessel growth was abolished in adiponectin (APN)-deficient mice. FGF21 administration also decreased neovascular lesions in two models of neovascular age-related macular degeneration, very-low-density-lipoproteinreceptor- deficient mice with retinal angiomatous proliferation and laser-induced choroidal neovascularization. FGF21 inhibited TNFα expression but did not alter Vegfa expression in neovascular eyes. These data suggest that FGF21 may be a therapeutic target for pathologic vessel growth in patients with neovascular eye diseases including retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration.

Keywords: retinal neovascularization, hypoxia, oxygen-induced retinopathy, choroidal neovascularization, FGF21

eTOC Blurb

Fu et al. find that FGF21 administration suppresses pathological ocular blood vessel growth in the retina and choroid. The inhibitory effects depend on adiponectin and TNFα, but are independent of VEGFA.

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Introduction

Pathological retinal neovessel growth is a major cause of vision loss in retinopathy of prematurity in premature infants, in macular telangiectasia, diabetic retinopathy and age-related macular degeneration in adults. Uncontrolled neovessel growth is driven by the need for oxygen and for energy substrates (Hellstrom et al., 2013; Joyal et al., 2016). Current treatments for retinal neovascularization have limitations. Although laser photocoagulation helps preserve central vision, it causes peripheral visual loss (Ciulla et al., 2003). In addition, anti-vascular endothelial growth factor (VEGF) drugs effectively treat neovascularization in some, but not all patients and there are safety concerns about the long-term effects including degeneration of normal blood vessels, neural retina and choroid (Arevalo, 2013; Cheung et al., 2012; Fernando Arevalo, 2013; Osaadon et al., 2014; Sato et al., 2012). Therefore, better therapeutic agents are needed for the effective treatment of vision-threatening neovascularization.

Fibroblast growth factors (FGFs) are generally thought to promote angiogenic responses (Cross and Claesson-Welsh, 2001). FGF21, one of the FGF family members, has been suggested as a pro-angiogenic factor in liver endothelial cells in vitro and in a mouse subcutaneous silicon-tube-implanted assay in vivo (Yaqoob et al., 2014). Here we found that FGF21 suppresses retinal and choroidal ocular pathologic angiogenesis in three different mouse models of disease. In humans, long-acting FGF21 analog administration increases circulating adiponectin (APN) in a dose-dependent manner (Gaich et al., 2013; Talukdar et al., 2016). In mice, FGF21 administration increases APN production to modulate glucose and lipid metabolism (Holland et al., 2013; Lin et al., 2013). Low circulating APN levels may contribute to the development of neovascular eye diseases in humans (Fu et al., 2016; Fu et al., 2015; Kaarniranta et al., 2012; Mao et al., 2012; Omae et al., 2015). APN administration inhibits retinal and choroidal neovascularization in rodents (Higuchi et al., 2009; Lyzogubov et al., 2012). We hypothesized that FGF21 may inhibit pathological retinal and choroidal angiogenesis in the eye and FGF21 administration could potentially improve neovascular eye diseases. We explored the role of FGF21 in 1) hypoxia-induced neovascular retinopathy (Smith et al., 1994); 2) retinal neovascularization driven by energy deficiency (Joyal et al., 2016); 3) laser-induced choroidal neovascularization (Gong et al., 2015).

Results and Discussion

FGF21 suppressed hypoxia-induced retinal neovascularization

Early vessel growth cessation or vessel loss in retinopathy of prematurity and diabetic retinopathy leads to hypoxia and nutrient deficits, which drives vessel overgrowth (Chen and Smith, 2007; Shin et al., 2014). To investigate the effects of FGF21 on pathologic neovessel growth under hypoxia, we administered FGF21 in mice with oxygen-induced retinopathy (Smith et al., 1994). After five-day exposure to 75% oxygen, mouse pups with their nursing dam were returned to room air. The oxygen exposure leads to vaso-obliteration in the central retina and the relatively avascular hypoxic retina induces neovascularization (Smith et al., 1994) extending from the retina into the vitreous at the boundary between vascular and non-vascularized areas (Connor et al., 2009). The mouse pups were intraperitoneally injected with native FGF21 or a long-acting FGF21 analog, PF-05231023 (Talukdar et al., 2016), or vehicle control for five days. Native FGF21 with a short half-life (0.4 hours) (Huang et al., 2013) did not affect neovascularization (Figure 1A) while administration of PF-05231023 with a biological half-life of 28 hours (Huang et al., 2013) significantly decreased neovascularization (Figure 1A). To confirm the role of FGF21 in retinal neovascularization, we examined the impact of FGF21 deficiency on oxygen-induced retinopathy comparing wild-type (Fgf21+/+) and knockout (Fgf21−/−) mice. Fgf21−/− mice had increased neovascularization (Figure 1B). Compared with the strong inhibition of pathologic neovessel formation by PF-05231023, the relatively smaller effects of native FGF21 and endogenous FGF21 deficiency on retinal neovascularization were possibly due to short half life of native FGF21 (0.4 hours) (Huang et al., 2013) and low endogenous FGF21 levels (about 658.3±66.4pg/ml in normal neonatal mouse serum detected by ELISA). To determine if FGF21 directly inhibits neovascularization, intra-vitreal injection of PF-05231023 reduced retinal neovascularization versus that of the vehicle-injected contralateral eye (Figure S1). FGF21 also promoted retinal revascularization through APN although APN may not be required for “basal” revascularization in OIR (Figure S2A–D). Improved revascularization will tend to decrease the stimulus for proliferative neovascularization.

Figure 1. FGF21 treatment decreased and FGF21 deficiency increased hypoxia-induced retinal neovascularization.

Figure 1

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Quantification of neovascularization in P17 retinal whole mounts after oxygen-induced retinopathy.

A) In retinas treated with native FGF21 (nFGF21), or 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., no significance.

B) In 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, 1mm. Data was presented as mean ± SEM. Fold of change was calculated comparing with the control group.

See also Figure S1 and S2

FGF21 exerts its effects through interaction with its receptor FGFR1 and co-receptor β-klotho (KLB) (Ding et al., 2012; Foltz et al., 2012; Suzuki et al., 2008). We found that FGF21 receptor 1, 2, 3, 4 and Klb mRNA were all expressed in the mouse retina (Figure 2A). Fgfr1 and fgfr3 were highly expressed in neovessels, and co-localized with APN (Figure S3A–B). PF-05231023 administration at P17 increased retinal Apn (Figure 2B), an important mediator of FGF21 effects on metabolic function (Holland et al., 2013; Lin et al., 2013). APN receptor agonist AdipoRon inhibited endothelial cell function in vitro (Figure S3C–D). To determine if APN also mediated the protection conferred by FGF21 against retinal neovascularization, we examined the retinal vasculature in APN knockout (Apn−/−) mice with or without PF-05231023 administration in oxygen-induced retinopathy. We found that APN deficiency worsened retinal neovascularization (Figure 2C) and lack of APN completely abolished the beneficial effects of PF-05231023 on reducing neovascularization in hypoxic retinas (Figure 2D). APN inhibits retinal neovascularization by decreasing the levels of tumor necrosis factor (TNF) α (Higuchi et al., 2009). We found that PF-05231023 suppressed Tnfα expression in neovascular WT retina but the suppression was abolished with APN deficiency (Figure 2E). In summary FGF21 inhibited pathological retinal neovessel growth possibly through targeting APN and reducing TNFα (Figure 2F), a key risk factor for oxygen-induced retinopathy (Kociok et al., 2006).

Figure 2. FGF21 suppressed retinal neovascularization via APN to reduce Tnfα.

Figure 2

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Quantification of neovascularization in P17 retinal whole mounts after oxygen-induced retinopathy.

A) In WT P17 normal retinas mRNA of FGF21 receptors Fgfr1, Fgfr2, Fgfr3, Fgfr4 and co-receptor β-klotho (Klb) (n = 6–8 pooled retinas per group).

B) In WT P17 hypoxic retinas, effect of PF-05231023 on retinal Apn (n = 6 pooled retinas per group). PF-05231023 was administered from P12–16.

C) In WT and Apn−/− mice (n = 17–20 retinas per group).

D) In 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 pseudo-colored white. Representative images are shown. Scale bar, 1mm. Data was presented as mean ± SEM. Unpaired t test. n.s., no significance. Fold of change was calculated.

See also Figure S3

Each year, over 15 million babies are born preterm with an incompletely vascularized retina at birth, which if normal vascularization does not occur postnatally, sets the stage for the progression to proliferative retinopathy (Hellstrom et al., 2013). Retinopathy of prematurity is a leading cause of blindness in children (Gilbert et al., 1997). We have previously demonstrated that low serum APN levels positively correlate with proliferative retinopathy in premature infants (Fu et al., 2015). Increasing circulating APN levels are associated with less retinal neovascularization in mice (Fu et al., 2015; Higuchi et al., 2010). We found that in mouse oxygen-induced retinopathy FGF21 administration increased retinal Apn levels and reduced Tnfα levels, and suppressed pathologic neovessel growth (Figure 2).

Diabetic retinopathy currently afflicts approximately 93 million people worldwide, and of those, 28 million have vision-threatening proliferative retinopathy (Abcouwer and Gardner, 2014). The levels of FGF21 in type 1 diabetes are lower than those in healthy controls (Xiao et al., 2012; Zibar et al., 2014). In streptozotocin-induced type 1 diabetic mice, a FGF21 analog reduces blood glucose levels with improved glucose uptake in brown adipose tissue (Kim et al., 2015). FGF21 prevents renal lipid accumulation, and attenuates renal dysfunction in type 1 diabetic mice (Zhang et al., 2013). In type 2 diabetes, serum FGF21 levels are higher in patients with retinopathy versus no retinopathy (Esteghamati et al., 2016; Lin et al., 2014). FGF21 treatment decreases body weight and improves the lipid profile (decreases triglycerides and increases HDL cholesterol levels) in type 2 diabetes patients, non-human primates and in obese rodents (Bernardo et al., 2015; Gaich et al., 2013; Schlein et al., 2016; Talukdar et al., 2016) suggesting that FGF21 might play a beneficial role in diabetes and diabetic complications, such as diabetic retinopathy. The mouse model of oxygen-induced retinopathy is commonly used to model hypoxia-induced neovascularization in proliferative diabetic retinopathy (Lai and Lo, 2013). Therefore, our findings also indicate that FGF21 may help prevent proliferative diabetic retinopathy. Further investigation is required in animal models of type 1 and type 2 diabetes to elucidate the role of FGF21 in early diabetic retinal neurovascular loss and late neovascularization regarding to the hyperglycemic aspect of diabetic retinopathy.

FGF21 administration protects against retinal neovascularization induced by energy-deficiency in Vldlr−/− mice

In addition to lack of oxygen, an inadequate fuel supply can also drive retinal neovascularization (Joyal et al., 2016). The absence of the very low density lipoprotein receptor (VLDLR) is associated with retinal angiomatous proliferation and choroidal neovascularization, similar to neovascularization seen with macular telangiectasia and late proliferative age-related macular degeneration (Engelbert and Yannuzzi, 2012; Lambert et al., 2016). In Vldlr−/− mice, abnormal blood vessels extend towards starved photoreceptors (Joyal et al., 2016) (Figure 3A). To assess whether FGF21 protects against metabolism-induced pathologic neovessel growth, we administered PF-05231023 intraperitoneally at 0.5mg/kg daily from P8 to P15 to Vldlr−/− mice. PF-05231023 administration attenuated the neovascular lesions (Figure 3A, B, C) and increased retinal Apn and decreased Tnfα (Figure 3D) in Vldlr−/− mice.

Figure 3. Exogenous FGF21 decreased retinal neovascularization in Vldlr−/− mice.

Figure 3

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A) 3D reconstruction of retinal neovessels extending from the outer plexiform layer (OPL) towards the retinal pigment epithelium (RPE). PF-05231023 (i.p. injected daily from P8-15) decreased retinal neovessel extension towards RPE seen with isolectin (red) stained vessels.

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, 1mm.

C) Number and area (size) of vascular lesions were quantified (n = 11–18 retinas per group).

D) In Vldlr−/− retinas, effect of PF-0523102 on Apn and Tnfα (n = 4–6 pooled retinas per group)

Data was presented as mean ± SEM. Unpaired t test. Fold of change was calculated.

FGF21 inhibited laser-induced choroidal neovascularization in mice

Choroidal neovascularization in which neovessels extend from the choriocapillaris into the subretinal space is vision-threatening in age-related macular degeneration. In the laser-induced choroidal neovascularization mouse model, laser burns disrupt Bruch’s membrane to induce choroidal neovessel growth (Ryan, 1979) (Figure 4A). We administrated 10mg/kg PF-05231023 every other day one week before and after the laser injury in 6–8-week-old C57B/6J mice. We found that PF-05231023 administration inhibited choroidal neovessel formation (Figure 4B). In the mouse choroidal neovascularization model, PF-05231023 also induced Apn and reduced Tnfα in the choroid-retina complex (Figure 4C).

Figure 4. FGF21 administration decreased choroidal neovascularization in mice.

Figure 4

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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 bar: 200μm (top); 50μm (bottom).

C) In neovascular choroid-retina complex, effect of PF-05231023 on Apn and Tnfα (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 was presented as mean ± SEM. Unpaired t test. n.s., no significance. Fold of change was calculated.

Neovascularization is a leading cause of vision loss in age-related macular degeneration and macular telangiectasia in the population over age 50 years (Heeren et al., 2014; Yonekawa et al., 2015). Metabolic alterations in retinal pigment epithelial cells and photoreceptors contribute to disease progression (Barron et al., 2001; Joyal et al., 2016). In Vldlr−/− mice which have an inadequate fuel supply driving retinal neovascularization, FGF21 administration reduced neovascular lesion formation. Our findings suggested that FGF21 administration might attenuate the development of metabolically driven retinal neovascularization, modeling macular telangiectasia and some aspects of neovascular age-related macular degeneration. Furthermore, in laser-induced choroidal neovascularization modeling inflammatory aspects of neovascular age-related macular degeneration (Parmeggiani et al., 2012), FGF21 also suppressed pathological choroidal angiogenesis in adult mice.

In summary, there is an unmet need for effective treatment of vision-threatening eye neovessel growth. We determined that FGF21 inhibited retinal and choroidal neovascularization mediated by APN in mice. APN suppresses TNFα transcription and mRNA stability in macrophages (Park et al., 2008; Wulster-Radcliffe et al., 2004). Inhibition of TNFα leads to decreased retinal and choroidal neovascularization (Kociok et al., 2006; Shi et al., 2006), possibly through increased endothelial cell sprouting (Hangai et al., 2006; Sainson et al., 2008). FGF21 promotes migration in HRMECs in vitro (Figure S3C). Different target system may be affected by FGF21 with opposite angiogenic responses. Our findings also suggest that FGF21 inhibitory effects on retinal and choroidal neovascularization are independent of VEGFA (Figure 4D). Given Lilly and Pfizer’s clinical data showing little to no effect of FGF21 on glycemic endpoints which has tempered enthusiasm for potential further development of FGF21 in the clinic for metabolic diseases, our data offers a phenotypic basis and some mechanistic insights into a new indication where FGF21 could be effective against retinopathy resulting in the next-generation standard of care for patients with pathological vascular proliferation in retinopathy of prematurity, diabetic retinopathy, macular telangiectasia and age-related macular degeneration. Further exploration of underlying mechanisms is desirable,

Experimental procedures

Detailed information is available in the Supplemental Experimental Procedures.

Study approval

All animal studies adhered to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee at Boston Children’s Hospital.

Mouse models of retinal/choroidal neovascularization

Oxygen-induced retinopathy: mice were exposed to 75% oxygen from P7 to P12. The mice were randomly assigned to either native FGF21 (5mg/kg, twice per day) or long-acting FGF21 (1mg/kg PF-05231023, a gift from Pfizer) or phosphate buffered saline (PBS). The mice were intraperitoneally injected daily from P12 to P16. For the intra-vitreal injection of PF-05231023 (0.5μl 10μg/μl stock per eye) was delivered using 10μl syringe with 35G needle (World Precision Instruments, Inc.) at P12. The contralateral eye was injected with vehicle control. At P17, retinal neovascularization was quantified (Connor et al., 2009; Smith et al., 1994).

Vldlr−/− mice

The mouse pups were treated with a long-acting FGF21 analogue (0.5mg/kg PF-05231023) or PBS from P8 to P15. At P16, neovascular lesions were quantified (Joyal et al., 2016).

Laser-induced choroidal neovascularization

Four laser burns were induced by a green Argon laser pulse with duration of 70ms and power of 240mW in 6–8-week-old C57BL/6J mice. A long-acting FGF21 analogue (10mg/kg PF-05231023) or PBS were intraperitoneally injected every other day one week before and after the laser photocoagulation induction. Lesion area was quantified (Gong et al., 2015).

Real-time PCR

RNA from retinas or choroid-retina complex was extracted and reverse-transcribed to cDNA. PCR was conducted for Fgfr1, Fgfr2, Fgfr3, Fgfr4, Klb, Apn, Vegfa, Dynamin2 and Tnfα. Cyclophilin A was used as internal control.

Statistics

All data were used except for low quality images that were insufficient for analysis. Data represent mean ± SEM. 2-tailed unpaired t-test or ANOVA with Bonferroni’s multiple comparison test was used for comparison of results as specified (Prism v5.0; GraphPad Software, Inc., San Diego, CA). Statistically significant difference was set at P ≤ 0.05. For phenotypic data, a dot represents each retina or choroid; for qPCR data, each dot represents a number of replicates from 4–6 pooled retinas.

Supplementary Material

1
2

Highlights.

  • FGF21 administration suppresses, and FGF21 deficiency worsens ocular neovessel growth in mice.

  • Suppression of retinal neovessel growth upon FGF21 administration is mediated by adiponectin

  • FGF21 decreased TNFα in neovascular retinas.

  • The inhibitory effects of FGF21 on eye neovascularization are independent of VEGFA.

Acknowledgments

We thank Drs. Steven Kliewer and David Mangelsdorf from the University of Texas Southwestern for providing Fgf21+/+ and Fgf21−/− mice. We thank Pfizer CVMED for providing native FGF21 and PF-05231023. LS is supported by NIH EY024864, EY017017, EY022275, P01 HD18655, Lowy Medical Research Institute, European Commission FP7 project 305485 PREVENT-ROP. ZF is supported by Knights Templar Eye Foundation and Bernadotte foundation. RL is supported by the German Research Foundation (DFG), Li2650/1-1.

Footnotes

S.T. is a current employee of Merck and was at the time the study was conducted. Merck has no role in any aspects of the work. The authors have declared that no conflict of interest exists.

Author contribution

L.E.H.S and S.T. are co-corresponding authors and both hold the responsibilities 1–7 indicated in the journal authorship policy. L.E.H.S is the lead contact. The contributions of each author are listed as below. Conceptualization, L.E.H.S, Z.F. and S.T.; Formal analysis, Z.F., S.T. and L.E.H.S.; Investigation, Z.F.; Writing-Original draft, Z.F.; Writing-Review & Editing, L.E.H.S., and S.T.; Resources, S.T.; Funding Acquisition, L.E.H.S.; Methodology, Z.F., Y.G., R.L., Z.W., C.H.L., S.S. M., S.B.B., N.J.S., T.W.F. and P.C.M.; Supervision, L.E.H.S, S.T. and A.H..

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NIHMS843807-supplement-1.pdf (541.7KB, pdf)
2
NIHMS843807-supplement-2.docx (11.2KB, docx)

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