Pemafibrate Prevents Retinal Pathological Neovascularization By

International Journal of Molecular Sciences

Article Pemaﬁbrate Prevents Retinal Pathological Neovascularization by Increasing FGF21 Level in a Murine Oxygen-Induced Retinopathy Model

1,2, 1,2, 1,2 1,2 3 Yohei Tomita †, Nobuhiro Ozawa †, Yukihiro Miwa , Ayako Ishida , Masayuki Ohta , Kazuo Tsubota 2,* and Toshihide Kurihara 1,2,* 1 Department of Ophthalmology, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan; [email protected] (Y.T.); [email protected] (N.O.); [email protected] (Y.M.); [email protected] (A.I.) 2 Laboratory of Photobiology, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan 3 Kowa Company, Ltd., Tokyo 160-8582, Japan; [email protected] * Correspondence: [email protected] (K.T.); [email protected] (T.K.); Tel.: +81-3-3353-1211 (K.T.); +81-3-3353-1211 (T.K.) These authors contributed equally to this work. † Received: 28 September 2019; Accepted: 21 November 2019; Published: 23 November 2019

Abstract: Large-scale clinical trials, such as the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) and the Action to Control Cardiovascular Risk in Diabetes (ACCORD) studies, have shown that the administration of fenofibrate, a peroxisome proliferator-activated receptor alpha (PPARα) agonist, suppresses the progression of diabetic retinopathy. In this paper, we reveal a therapeutic effect of a selective PPARα modulator (SPPARMα), pemafibrate, against pathological angiogenesis in murine models of retinopathy. Oxygen-induced retinopathy (OIR) was induced in C57BL/6J mice by exposure to 85% oxygen from postnatal day eight (P8) for 72 h. Vehicle, pemafibrate or fenofibrate was administrated by oral gavage from P12 to P16 daily. Administration of pemafibrate, but not fenofibrate, significantly reduced pathological angiogenesis in OIR. After oral pemafibrate administration, expression levels of downstream PPARα targets such as acyl-CoA oxidase 1 (Acox1), fatty acid binding protein 4 (Fabp4), and fibroblast growth factor 21 (Fgf21) were significantly increased in the liver but not in the retina. A significant increase in plasma FGF21 and reduced retinal hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor A(Vegfa) were also observed after this treatment. In an in vitro HIF-luciferase assay, a long-acting FGF21 analogue, but not pemafibrate, suppressed HIF activity. These data indicate that SPPARMα pemafibrate administration may prevent retinal pathological neovascularization by upregulating FGF21 in the liver.

Keywords: diabetes retinopathy; selective peroxisome proliferator-activated receptor alpha modulator (SPPARMα); ﬁbroblast growth factor 21 (FGF21); hypoxia-inducible factor (HIF); vascular endothelial growth factor (VEGF)

1. Introduction Diabetic retinopathy (DR) and age-related macular degeneration (AMD) are major causes of vision impairment worldwide. The number of diabetic patients increases every year, so the prevention of DR onset has become an increasingly important issue in many countries [1]. These diseases are characterized by pathological angiogenesis [1,2]. In the last few decades, anti-vascular endothelial growth factor (VEGF) therapy has been established to preserve and maintain the visual function in these neovascular retinal diseases [3–5]. As a pathophysiological mechanism of DR, retinal ischemia

Int. J. Mol. Sci. 2019, 20, 5878; doi:10.3390/ijms20235878 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 5878 2 of 11 has been shown to produce angiogenic and inflammatory agents including VEGF, which induce the activity of inflammatory leukocytes, and later cause pathological angiogenesis [6]. Anti-VEGF therapy is now being applied to diabetic macular edema, as well as proliferative retinopathy. However, anti-VEGF therapy still faces several difficulties, such as affordability, difficulty with visual acuity improvement, and potential for geographic atrophy after prolonged exposure to treatment [7]. Therefore, developing an alternative therapeutic treatment is necessary. Besides the pathological role of retinal ischemia in DR, metabolic changes in the retinal tissues due to hypoxic conditions were also reported and may contribute to the onset of AMD [8]. Lipid deposition has also been shown to play a role in AMD pathogenesis. Throughout aging, lipid metabolites deposited and peroxidized under retinal pigment epithelial (RPE) cells may cause chronic inflammation and lead to the onset of AMD [9,10]. Therefore, the regulation of lipid metabolism is important to consider when addressing this disease. According to two large randomized controlled trials, the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study [11] and the Action to Control Cardiovascular Risk in Diabetes (ACCORD) study [12], a fibric acid derivative, fenofibrate, inhibits the progression of DR and other microvascular endpoints in patients with type 2 diabetes. Fenofibrate is primarily used as a hypolipidemic agent, which largely reduces triglyceride levels and increases high-density lipoprotein (HDL)-cholesterol levels, by activating peroxisome proliferator-activated receptor alpha (PPARα). Although activation of PPARα by fenofibrate has been shown to suppress pathological angiogenesis, precise mechanisms of a PPARα activator fenofibrate in DR are not yet fully understood [13]. In recent years, a selective PPARα modulator (SPPARMα) pemafibrate (Parmodia®, K-877, Kowa, Tokyo, Japan) was approved in Japan in July 2017 as a therapeutic drug against hyperlipidemia. Pemafibrate reduces serum triglycerides (TGs) and increases the HDL cholesterol in a similarly way to fenofibrate [14]. Unlike the renal excretion of fenofibrate, pemafibrate is metabolized in the liver, and consequently can be used in patients with mild renal impairment. In addition, pemafibrate has been shown to activate PPARα more specifically than fenofibrate [15]. However, the therapeutic effects of pemafibrate in ocular diseases like AMD and DR have not yet been reported. Pemafibrate has been reported to activate PPARα in hepatocytes resulting in an increase of plasma fibroblast growth factor 21 (FGF21) levels [16]. FGF21 is a secreted protein composed of 209 amino acids that was first reported in 2000 [17]. Systemic administration of FGF21 has favorable effects on glucose and lipid metabolism in mice and improves the level of blood lipids in type 2 diabetic patients [18]. Recently, a long-acting FGF21 was shown to suppress pathological neovascularization in the retina and choroid [19], thereby exerting a protective effect on the neural retina [20]. In this study, using the mouse oxygen-induced retinopathy (OIR) model, which is the best characterized and widely used in vivo retinal neovascularization model [21,22], we examined whether the administration of pemafibrate has an inhibitory effect on pathological angiogenesis. We explored the mechanism underlying the induction of FGF21 expression. Through the results of this study we wanted to uncover future potential therapeutic reagents to address prevalent retinopathies.

2. Results

2.1. Antiangiogenic Effect of Oral Administration of Pemafibrate on the Retina of OIR To investigate the effect of pemafibrate on angiogenesis, we used the retinas of OIR model mice (Figure1A–F). We found no significant di fferences in body weight among the groups at postnatal day 12 (P12) or P17 (Figure1G). There were no significant di fferences in vaso-obliteration (VO) among the groups (p = 0.19) (Figure1H). The neovascular tufts (NV) area in the pemafibrate group was significantly decreased compared with the vehicle group; however, no significant changes were found between the fenofibrate and the vehicle groups (Figure1I). These data indicate that oral administration of pemafibrate prevents pathological but not physiological retinal neovascularization. Int. J. Mol. Sci. 2019, 20, 5878 3 of 11 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 12

FigureFigure 1.1. PemafibratePemafibrate hashas anan anti-angiogenicanti-angiogenic eeffectffect inin thethe retina.retina. ((AA––FF)) RepresentativeRepresentative retinalretinal imagesimages ofof the each each oxygen-induced oxygen-induced retinopathy retinopathy (OIR) (OIR) model model mice mice (red, (red, neovascular neovascular tufts tufts(NV); (NV); yellow, yellow, vaso- vaso-obliterationobliteration (VO)), (VO)), scale scalebar: 500 bar: μm. 500 (Gµm.) The (G change) The change in the body in the weig bodyht weightamong amongthe groups the groups(day 12 (day(P12) 12 and (P12) P17, andn = 6). P17, (H)n Quantification= 6). (H) Quantification of VO area with of VOeach area group with (P17, each n = group10,11). ( (P17,I) Quantificationn = 10,11). (ofI) QuantificationNV area with each of NV group area (P17, with n each = 10 group,11). Note (P17, thatn =oral10,11). administration Note that of oral pemafibrate administration prevents of pemafibratepathological preventsbut not pathologicalretinal neovascularization but not retinal. The neovascularization. data were analyzed The by data 1-way were ANOVA analyzed and by 1-way ANOVA and Tukey’s multiple comparison test and are expressed as mean standard error (SE). Tukey’s multiple comparison test and are expressed as mean ± standard error± (SE). ** p < 0.01. n.s., **notp < significant.0.01. n.s., not significant. 2.2. Pemafibrate Directly Acts in the Liver and Promotes Expression of Factors Downstream of PPARα 2.2. Pemafibrate Directly Acts in the Liver and Promotes Expression of Factors Downstream of PPARα Next, we explored the primary target organ of the drug. In the retina, no significant differences Next, we explored the primary target organ of the drug. In the retina, no significant differences occurred in expression between the pemafibrate and the vehicle groups for genes downstream of PPARα, occurred in expression between the pemafibrate and the vehicle groups for genes downstream of including acyl-CoA oxidase 1 (Acox1), fatty acid binding protein 4 (Fabp4), and Fgf21 (Figure2A–C). PPARα, including acyl-CoA oxidase 1 (Acox1), fatty acid binding protein 4 (Fabp4), and Fgf21 (Figure In contrast, the mRNA expression levels of these genes were significantly higher in the liver of the 2A–C). In contrast, the mRNA expression levels of these genes were significantly higher in the liver pemafibrate group compared with the vehicle group (Figure2D–F). These data suggest that oral of the pemafibrate group compared with the vehicle group (Figure 2D–F). These data suggest that administration of pemafibrate directly affects the liver but not the retina. oral administration of pemafibrate directly affects the liver but not the retina.

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Figure 2.FigurePemafibrate 2. Pemafibrate stimulates stimulates peroxisome peroxisome proliferator-activated proliferator-activated receptor receptor alpha alpha (PPAR (PPARα) downstreamα) downstream gene expression in the liver but not in the retina. (A–C) The mRNA expression levels of α gene expression in the liver but not in the retina. (A–C) The mRNA expression levels of PPAR downstreamPPARα genesdownstream including genes includ acyl-CoAing acyl-CoA oxidase oxidase 1 (Acox1) 1 (Acox1),, fatty fatty acid acid binding binding protein protein 4 (Fabp4), 4 (Fabp4), Figureand fibroblast 2. Pemafibrate growth factor stimulates 21 (Fgf21) peroxisome in the retina prolif (P17,erator-activated n = 7,8) and (D– Freceptor) in the liver alpha (D– F(PPAR; P17, nα )= and fibroblast growth factor 21 (Fgf21) in the retina (P17, n = 7,8) and (D–F) in the liver (D–F; downstream4) in OIR model gene mice. expression Note that in the oral liver administrati but not inon the of retina. pemafibrate (A–C) Theincreased mRNA the expression targeted levelsgenes ofin P17, n =PPARthe4) liver inα downstream OIR but modelnot in thegenes mice. retina. includ Note Theing thatdata acyl-CoA oralwere administrationoxidaseanalyzed 1 (usingAcox1) Student’s, offatty pemafibrate acid t-test binding and proteinincreasedare expressed 4 (Fabp4), the as targeted genes inandmean the fibroblast liver± SE. *** but growth p not< 0.001; in factor the ****retina. 21p < ( Fgf21)0.0001. The in n.s., datathe not retina were significant. (P17, analyzed n = 7,8) usingand (D Student’s–F) in the livert-test (D and–F; P17, are n expressed = as mean4) inSE. OIR *** modelp < 0.001;mice. Note **** thatp < oral0.0001. administrati n.s., noton significant. of pemafibrate increased the targeted genes in 2.3. Pemafibratethe± liver but Increases not in the Plasma retina. FGF The 21data Concentrat were analyzedion and using Suppresses Student’s Expression t-test and of areVegfa expressed in the Retina as 2.3. Pemafibratemean Increases± SE. *** p < Plasma 0.001; **** FGF p < 0.0001. 21 Concentration n.s., not significant. and Suppresses Expression of Vegfa in the Retina We focused on FGF21 as its mRNA expression was increased in the liver after pemafibrate We2.3.administration. focused Pemafibrate on Increases FGF21The plasma Plasma as itsFGF21 FGF mRNA concentration21 Concentrat expressionion was and significantly was Suppresses increased Expression elevated in thein of theVegfa liver pemafibrate in the after Retina pemafibrate and administration.fenofibrate The group plasma (P13) compared FGF21 concentration with the contro wasl group significantly (Figure 3A). elevatedThe mRNA in expression the pemafibrate level and of Fgf21We focusedwas significantly on FGF21 increasedas its mRNA in the expression pemafibrate was notincreased fenofibrate in the (Figure liver after 3B). pemafibrateThe mRNA fenofibrateadministration.expression group level (P13) The of comparedVegfa plasma significantly FGF21 with concentration thedecreased control in was groupthe significantlypemafi (Figurebrate 3andelevatedA). fenofibrate The in mRNA the pemafibrategroup expression compared and level of Fgf21 wasfenofibratewith significantly the vehicle group group increased(P13) (Figurecompared in 3C). the with These pemafibrate the data contro suggel notgroupst that fenofibrate (Figure elevated 3A). plasma (Figure The mRNA FGF213B). The mayexpression mRNA be involved level expression level ofofinVegfa theFgf21 inhibitionsignificantly was significantly of Vegfa decreased within increased the retina. in in the the pemafibrate pemafibrate not and fenofibrate fenofibrate (Figure group 3B). compared The mRNA with the vehicleexpression group (Figure level of3 C).Vegfa These significantly data suggest decreased that in the elevated pemafibrate plasma and fenofibrate FGF21 may group be compared involved in the with the vehicle group (Figure 3C). These data suggest that elevated plasma FGF21 may be involved inhibition of Vegfa within the retina. in the inhibition of Vegfa within the retina.

Figure 3. Pemafibrate and fenofibrate increases the FGF21 concentration in the plasma and suppress Vascular endothelial growth factor A (Vegfa) expression in the retina. (A) The plasma concentration

of FGF21 in OIR model mice at P13 (n = 2,3). (B) The mRNA expression of Fgf21 in the liver in OIR FigureFigure 3.modelPemafibrate 3.mice Pemafibrate at P13 and (n =and fenofibrate3). fenofibrate(C) The mRNA increases increases expression the FGF FGF21 of21 Vegfa concentration concentration in the retina in inthe inOIR plasma the model plasma and mice suppress andat P13 suppress VascularVascular(nendothelial = 5,6). endothelialNote that growth oral growth administration factor factor A (AVegfa (Vegfa of) pemafi expression) expressionbrate and inin the fenofibrate retina. retina. ( Aincreased ()A The) The plasma the plasma plasma concentration concentration level of of FGF21 inofFGF21 FGF21 OIR and model in suppressedOIR mice model at themice P13 retinal (atn P13= 2,3).expression (n = (2,3).B) The (ofB) VegfaThe mRNA mRNA. A significant expression expression upregulation of ofFgf21 Fgf21in inof the theFgf21 liver mRNA in in OIR OIR in model model mice at P13 (n = 3). (C) The mRNA expression of Vegfa in the retina in OIR model mice at P13 mice atthe P13 liver (n = was3). observed (C) The in mRNA the pemafibrate expression group, of butVegfa notin in thefenofibrate retina ingroup. OIR The model data micewere analyzed at P13 ( n = 5,6). (n = 5,6). Note that oral administration of pemafibrate and fenofibrate increased the plasma level of Note that oral administration of pemafibrate and fenofibrate increased the plasma level of FGF21 and FGF21 and suppressed the retinal expression of Vegfa. A significant upregulation of Fgf21 mRNA in suppressedthe liver the was retinal observed expression in the pemafibrate of Vegfa. group, A significant but not in upregulation fenofibrate group. of Fgf21 The datamRNA were analyzed in the liver was observed in the pemafibrate group, but not in fenofibrate group. The data were analyzed by 1-way

ANOVA and Tukey’s multiple comparison tests and are expressed as mean SE. * p < 0.05, ** p < 0.01, ± **** p < 0.0001. n.s., not signiﬁcant. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 5 of 12

by 1-way ANOVA and Tukey’s multiple comparison tests and are expressed as mean ± SE. * p < 0.05, Int. J.** Mol. p < Sci. 0.01,2019 ****, 20 p, 5878< 0.0001. n.s., not significant. 5 of 11

2.4. Oral Administration of Pemafibrate Inhibits the Retinal Expression of HIF-1α 2.4. Oral Administration of Pemafibrate Inhibits the Retinal Expression of HIF-1α To evaluate the mechanism underlying the suppression of Vegfa expression, we focused on hypoxia-inducibleTo evaluate the factor-1 mechanismα (HIF-1 underlyingα), which the is suppressiona transcription of Vegfa factorexpression, controlling we Vegfa focused gene on expression.hypoxia-inducible Using immunohistochemistry, factor-1α (HIF-1α), which we is detected a transcription a decrease factor in controlling the expressionVegfa ofgene HIF-1 expression.α in the retinalUsing immunohistochemistry,inner layer in the OIR model we detected mouse pemafi a decreasebrate ingroup the expression(Figure 4). ofThese HIF-1 dataα indemonstrate the retinal thatinner oral layer administration in the OIR model of pemafibrate mouse pemafibrate suppresses group retinal (Figure HIF-14α). expression These data in demonstrate OIR model thatmice. oral administration of pemafibrate suppresses retinal HIF-1α expression in OIR model mice.

Figure 4. The systemic administration of pemafibrate inhibits hypoxia-inducible factor-1α (HIF-1α) Figurein the retina. 4. The Ocularsystemic cross-sections administration of OIRof pemafibrate model mice inhibits (P17) injected hypoxia-inducible with pemafibrate factor-1 andα (HIF-1 vehicle,α) inand the normoxia retina. Ocular mice werecross-sections immune-stained of OIR model with antibodies mice (P17) specific injected for with HIF-1 pemafibrateα (green). and Nuclei vehicle, were andcounterstained normoxia mice with were DAPI immune-stained (blue). scale bar: with 500 µantibodiesm. specific for HIF-1α (green). Nuclei were counterstained with DAPI (blue). scale bar: 500 μm. 2.5. Inhibitory Effect of FGF21 on HIF Activity in Vitro 2.5. InhibitoryTo examine Effect HIF of inhibitoryFGF21 on eHIFffects Activity of pemafibrate in Vitro and a long-acting FGF21 molecule (PF-05231023), a HIF luciferase assay was performed in vitro. Pemafibrate or PF-05231023 was added to the 661W cell To examine HIF inhibitory effects of pemafibrate and a long-acting FGF21 molecule (PF- line, which has properties of both retinal ganglion and photoreceptor cells [23], whereas pseudohypoxia 05231023), a HIF luciferase assay was performed in vitro. Pemafibrate or PF-05231023 was added to was induced with CoCl . PF-05231023 (0.2 and 2 nM) significantly inhibited HIF activity compared with the 661W cell line, which2 has properties of both retinal ganglion and photoreceptor cells [23], whereas CoCl2 alone (Figure5A). In contrast, administration of pemafibrate with CoCl 2 showed no inhibitory pseudohypoxia was induced with CoCl2. PF-05231023 (0.2 and 2 nM) significantly inhibited HIF effect on HIF activity (Figure5B). These data suggest that systemic administration of pemafibrate activity compared with CoCl2 alone (Figure 5A). In contrast, administration of pemafibrate with might indirectly inhibit retinal HIF activity by elevating the plasma FGF21 concentration. CoCl2 showed no inhibitory effect on HIF activity (Figure 5B). These data suggest that systemic administration of pemafibrate might indirectly inhibit retinal HIF activity by elevating the plasma FGF21 concentration.

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(A) (B)

Figure 5. AA long-acting FGF21 FGF21 molecule molecule but but not not pemafibrat pemafibratee has has an an inhibitory inhibitory effect effect on on HIF activity inin vitrovitro.. HIF-reporter HIF-reporter luciferase luciferase assay assay was performed with 661W cells. ( (AA)) A A long-acting long-acting FGF21 FGF21 molecule (PF-05231023; 0.2 and 2 nM) and ( B)) pemafibrate pemafibrate (10 (10 and and 100 100 nM) nM) were were added added together together with with

CoCl22. .Note Note that that PF-05231023 PF-05231023 but but not not pemafibrate pemafibrate sign significantlyificantly inhibited inhibited HIF HIF activity activity induced induced by by CoCl22.. The The data data were were analyzed analyzed by by one-way one-way ANOVA ANOVA an andd Tukey’s Tukey’s multiple comparison tests tests and are expressedexpressed as mean ± SE.SE. * * pp << 0.05,0.05, ** ** pp << 0.01,0.01, **** **** p p< <0.0001).0.0001). n.s., n.s., not not significant. significant. ± 3. Discussion Discussion InIn the the current current study, study, we revealed that that oral oral administration administration of of a a selective selective PPAR PPARαα modulator,modulator, pemafibrate,pemafibrate, inhibitedinhibited pathological pathological angiogenesis angiogenesis in the in retinathe reti of OIRna of model OIR micemodel (Figure mice1 ).(Figure A significant 1). A significantincrease in increase PPARα target in PPAR geneα expressiontarget gene in expression the liver (Figure in the2 liver) and (Figure an elevation 2) and of an the elevation plasma FGF21 of the plasmaconcentration FGF21 (Figure concentration3) was observed (Figure after3) was systemic observed administration after systemic of pemafibrate,administration which of pemafibrate, is consistent whichwith a is previous consistent report with of a pemafibrate previous report increasing of pemafibrate FGF21 expression increasing in FGF21 patients expression with type in 2 diabetespatients with type hypertriglyceridemia 2 diabetes with hypertriglyceridemia and in high fat diet mice and [in24 high,25]. fat The diet authors mice [24,25]. showed The that authors the elevation showed of thatFGF21 the after elevation pemafibrate of FGF21 administration after pemafibrate was not administration observed in PPARwas notα knockout observed mice in PPAR [25]. α knockout mice Recently[25]. a long-acting FGF21 molecule, PF-05231023, was reported to have anti-angiogenic activityRecently in the a retina long-acting of OIR modelFGF21 micemolecule, [19]. In PF-05231023, this report, theywas showedreported that to increasedhave anti-angiogenic adiponectin activityexpression in the after retina PF-05231023 of OIR model administration mice [19]. produced In this report, anti-angiogenic they showed effects that through increased the inhibitionadiponectin of expressiontumor necrosis after factorPF-05231023 (TNF)- αadministration. However, in produced our experiment, anti-angiogenic we found effects no di throughfference the in the inhibition mRNA ofexpression tumor necrosis level of factor adiponectin (TNF)-α and. However, TNF-α betweenin our experiment, the vehicle we and found pemafibrate no difference groups, in the in themRNA OIR expressionmodel (data level not shown).of adiponectin and TNF-α between the vehicle and pemafibrate groups, in the OIR modelA (data study not reported shown). that fenofibrate prevents pathological neovascularization in the rat OIR model by suppressingA study reported HIF and that VEGF fenofibrate [26]. HIF prevents plays an pathol importantogical role neovascularization in maintaining tissue in the homeostasis rat OIR model after byexposure suppressing to hypoxic HIF conditionsand VEGF and[26]. other HIF stressplays stimulian important [27,28]. role Thus, in wemaintaining also focused tissue on the homeostasis HIF/VEGF aftersystem exposure as a potential to hypoxic pathway conditions activated and by other pemafibrate. stress stimuli In addition, [27,28]. HIF Thus, stabilization we also focused induces on target the HIF/VEGFgene expression, system such as a aspotentialVegfa and pathway pyruvate activate dehydrogenased by pemafibrate. lipoamide In addition, kinase isozymeHIF stabilization 1 (Pdk1), inducesfor anaerobic target metabolism gene expression, [29,30 ].such In fact, as Vegfa we have and previouslypyruvate dehydrogenase found that multiple lipoamide HIF-responsive kinase isozyme genes 1including (Pdk1), forerythropoietin anaerobic, angiopoietin-2metabolism [29,30]., and Pdk1, In fact,in addition we have to Vegfa,previouslywere upregulatedfound that inmultiple the retina HIF- of responsiveOIR model micegenes [31 including]. In this current erythropoietin experiment,, angiopoietin-2 administration, and of Pdk1, pemafibrate in addition suppressed to Vegfa, the mRNA were upregulatedexpression of inVegfa the inretina the retinaof OIR of model P13 OIR mice model [31] mice. In this (Figure current3). Pemafibrate experiment, suppressed administration HIF-1 ofα pemafibrateexpression in suppressed the inner retinathe mRNA of OIR expression model mice of Vegfa (Figure in4 the). From retina the of resultsP13 OIR of model the HIF mice luciferase (Figure 3).assay, Pemafibrate PF-05231023 suppressed suppressed HIF-1 HIFα expression activity inretinal in the inner photoreceptor retina of cells.OIR model In contrast, mice (Figure pemafibrate 4). From did thenot results show a of HIF the inhibitory HIF luciferase effect. assay, Taken PF-0523102 together, the3 suppressed direct target HIF organ activity of systemic in retinal administration photoreceptor of cells.pemafibrate In contrast, is presumably pemafibrate the did liver, not which show cana HIF produce inhibitory FGF21 effect. to suppress Taken together, HIF activity, the direct and thereby target organinhibit of retinal systemic pathological administration angiogenesis of pemafibrate (Figure6 ).is presumably the liver, which can produce FGF21 to suppress HIF activity, and thereby inhibit retinal pathological angiogenesis (Figure 6).

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In the current study, pemafibrate, but not fenofibrate, protected against pathological retinal neovascularizationInt. J. Mol. Sci. (Figure2019, 20, x FOR1). PEER Studies REVIEW have shown that fenofibrate administration with a7 of similar 12 dose to the currentIn study the current had anstudy, inhibitory pemafibrate, effect but againstnot fenofibrate, neovascularization protected againstin pathological OIR model retinal mice [32,33]. The disagreementneovascularization in results (Figure may 1). beStudies due have to di shownfferences that fenofibrate between administration intervention with routes a similar or dose experimental protocols.to Both the current chemicals study showed had an inhibitory similar trendseffect agai ofnst increased neovascularization levels of in plasma OIR model FGF21 mice and [32,33]. upregulation of Fgf21 expressionThe disagreement in the in liver, results although may be due the to differen resultces of fenofibratebetween intervention administration routes or experimental had high variation protocols. Both chemicals showed similar trends of increased levels of plasma FGF21 and (Figure3).upregulation Although of both Fgf21 fenofibrate expression in and the liver, pemafibrate although the a ff resultect PPAR of fenofibrateα, the eadministrationffects are di hadfferent. First, fenofibratehigh induces variation not (Figure only 3). PPAR Althoughα but both also fenofibrate PPAR andδ, whereaspemafibrate pemafibrate affect PPARα, inducesthe effects onlyare PPARα, by bindingdifferent. to the entireFirst, fenofibrate cavity region induces of not ligand-binding only PPARα but pocket also PPAR [34δ]., whereas Second, pemafibrate PPARα agonists induces have the potential toonly trigger PPAR diα,ff byerent binding biological to the entire responses cavity region via the of sameligand-binding receptor pock [35et]. [34]. As such, Second, these PPAR findingsα may agonists have the potential to trigger different biological responses via the same receptor [35]. As indicate thatsuch, pemafibrate these findings is moremay indicate powerful that thanpemafibr fenofibrateate is more for powerful the prevention than fenofibrate of neovascularization. for the A meta-analysisprevention of of RCTs neovascularization. showed that A in meta-analysis humans, pemafibrate of RCTs showed exhibits that in significantlyhumans, pemafibrate fewer adverse events and,exhibits an increase significantly in creatinine fewer adverse levels events was and, significantly an increase lowerin creatinine than fenofibratelevels was significantly [36]. These may be the reasonslower why than pemafibrate fenofibrate [36]. and These fenofibrate may be the have reasons di whyfferent pemafibrate effects forand thefenofibrate retina. have different effects for the retina.

Figure 6. TheFigure suppressive 6. The suppressive mechanism mechanism of pathological of pathological retinalretinal angiogenesis angiogenesis by systemic by systemic administration administration of of pemafibrate. Systemic selective PPARα modulator (SPPARMα) pemafibrate administration α α pemaﬁbrate.upregulates Systemic PPAR selectiveα target PPARgenes includingmodulator Fgf21 in (SPPARM the liver, but) not pemaﬁbrate in the retina. administration The increased level upregulates PPARα targetof the genes plasma including FGF21 concentrationFgf21 in resulting the liver, in butthe downregulation not in the retina. of Vegfa The via increased HIF inactivation, level may of the plasma FGF21 concentrationbe a possible mechanism resulting for in prevention the downregulation of retinal pathological of Vegfa neovascularization.via HIF inactivation, may be a possible mechanism for prevention of retinal pathological neovascularization. 4. Materials and Methods 4. Materials and Methods 4.1. Ethics Statement 4.1. Ethics StatementAll animal studies adhered to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, from the National Institutes of All animalHealth studies(NIH) guidelines adhered for to work the Associationwith laboratory for animals, Research “Animal in Vision Research: and Reporting Ophthalmology of in vivo Statement for the UseExperiments of Animals (ARRIVE)” in Ophthalmic guidelines, and and Vision were approved Research, (#16017-1) from the on National25 October Institutes2017 by the ofEthics Health (NIH) guidelinesCommittee for work for with Animal laboratory Research of animals, Keio University “Animal School Research: of Medicine Reporting (Tokyo, Japan). of in vivo Experiments (ARRIVE)” guidelines, and were approved (#16017-1) on 25 October 2017 by the Ethics Committee for Animal Research of Keio University School of Medicine (Tokyo, Japan).

4.2. Mice C57BL/6J mice were obtained from CLEA Japan, Inc. OIR was induced in the mice by exposure to 85% oxygen from postnatal day 8 (P8) for 72 h. Pemaﬁbrate (0.3 mg/kg/day; Kowa, Tokyo, Japan), Int. J. Mol. Sci. 2019, 20, 5878 8 of 11 fenoﬁbrate (10 mg/kg/day; Sigma-Aldrich, St. Louis, MO, USA) and vehicle (methyl cellulose: Shin-Etsu Chemical, Tokyo, Japan) were fed via oral gavage from P12 to P16 daily. Mice under normoxia with vehicle were also prepared (NOX group). At P17, the vaso-obliteration (VO) and neovascular tuft (NV) areas were evaluated on the wholemount retinae with iso-lectin B4 staining, as previously described [21].

4.3. Real-Time PCR The expression levels of various genes were measured in the retina and the liver from the sacriﬁced animals at P13 and P17. The total mRNA of the retina was extracted from the mouse retinal samples using RNeasy Plus Mini kit (Qiagen, Velno, Netherlands), and reverse-transcribed using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). Real-time RT-PCR was performed using THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan). Data were analyzed with StepOne, Software version 2.3 (Applied Biosystems, Waltham, MA, USA). The total mRNA of the liver was extracted from the mouse liver homogenate following the manufacturer’s instructions using ISOGEN II (Nippon Gene, Tokyo, Japan). Reverse transcription of the extracted mRNA was performed using High-capacity cDNA Reverse Transcription Kits (Life Technologies, Carlsbad, CA, USA) in accordance with their instructions. Fast SYBR® Green Master Mix (Applied biosystems, Waltham, MA, USA) was used as the reagent, and the Fast Real-Time PCR System (7900HT, Applied Biosystems, Waltham, MA, USA) was used for quantitative PCR. The primer sequences for gene expression analysis were: mouse β-actin: forward, 50-GGAGGAAGAGGATGCGGCA-30, reverse, 50-GAAGCTGTGCTATGTTGCTCTA-30, mouse Fgf21: forward, 50-ACAGCCATTCACTTTGCCTGAGC-30, reverse, 50-GGCAGCTGGAATTGTGTTCTGACT-30, mouse Acox1: forward, 50-TCTTCTTGAGACAGGGCCCAG-30, reverse, 50-GTTCCGACTAGCCAGGCATG-30, mouse Fabp4: forward, 50-CCGCAGACGACAGGA-30, reverse, 50-CTCATGCCCTTTCATAAACT-30, mouse Vegfa: forward, 50-CCCTCTTAAATCGTGCCAAC-30, reverse, 50-CCTGTCCCTCTCTCTGTTCG-30. β-actin was used as an internal control.

4.4. Measurement of Plasma FGF21 Concentrations of FGF21 in the plasma was measured at P13 and P17. The plasma FGF21 levels were measured with a microplate reader (VersaMax, Molecular Devices, San Jose, CA, USA) using the FGF-21 ELISA kit (#RD291108200R, BioVendor Laboratory Medicine, Brno, Czech Republic) according to the manufacturer’s protocol.

4.5. Immunohistochemistry Immunohistochemistry (IHC) for the retina was performed as previously described [37]. The primary antibody for HIF-1α was established by immunizing puriﬁed fusion proteins encompassing amino acids 416 to 785 of mouse HIF-1α, into guinea pigs [37]. Alexa Fluor 488 conjugated IgG (Life Technologies) was used as the secondary antibody. Nuclei were stained with DAPI (Fluoromount-G, SouthernBiotech, Birmingham, AL, USA). The images were captured with a ﬂuorescence microscope (BZ 9000, Keyence, Osaka, Japan).

4.6. HIF-Luciferase Assay To monitor HIF transcriptional activity, the mouse 661W cell line, which has aspects of both photoreceptor and retinal ganglion cells, was transfected with a HIF-luciferase reporter gene construct (Cignal Lenti HIF reporter, Qiagen). The luciferase construct encodes the firefly luciferase gene under the control of a hypoxia-related element that connects to HIF. CMV-Renilla luciferase was also co-transfected into these cells as an internal control. q before luminescence was measured, HIF activation of normal oxygen pressure was induced by administering cobalt chloride (CoCl2, 200 µM, cobalt (II) chloride Int. J. Mol. Sci. 2019, 20, 5878 9 of 11 hexahydrate, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) to the cells. To evaluate the inhibitory effect of a long-acting FGF21 molecule (PF-05231023, MedKoo Biosciences, Morrisville, NC, USA) and pemafibrate on HIF activation, these two drugs were added at the same time as CoCl2 administration. Luminescence was measured with Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA). The suppressive effect of pemafibrate and PF-05231023 against HIF activation was evaluated 24 h after the induction.

4.7. Statistical Analysis All data are expressed as means standard error. Statistical signiﬁcance was calculated ± using Student’s t-test or 1-way ANOVA and Tukey’s multiple comparison tests with Prism 6 (GraphPad Software, San Diego, CA, USA).

5. Conclusions SPPARMα pemaﬁbrate alleviated pathological neovascularization in the retina by attenuating the HIF/VEGF pathway through the induction of Fgf21 in the liver, but not in the retina. Although further investigations are required to better understand the mechanism underlying this phenomenon, the current results indicate that oral administration of pemaﬁbrate may be a promising therapeutic strategy to treat neovascular retinal diseases.

Author Contributions: Y.T., N.O., Y.M., K.T., and T.K. designed all experiments; Y.T., N.O., Y.M., A.I., and M.O. performed the experiments; Y.T., N.O., A.I., Y.M., and M.O. acquired and analyzed the data; Y.T. and T.K. wrote the manuscript. All authors read and approved the final manuscript. Funding: This work is supported by Grants-in-Aid for Scientific Research (KAKENHI, numbers 15K10881 and 18K09424) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to T. K. and grants from Kowa Life Science Foundation to Y.T. Acknowledgments: We are grateful for Kowa, Co., Ltd. for providing pemafibrate. We thank H. Torii, S. Ikeda, Y. Hagiwara, K. Mori, Y. Katada, H. Kunimi, E. Yotsukura, M. Ibuki, C. Shoda, Y. Wang, and K. Takahashi at the Laboratory of Photobiology and L.E.H. Smith, Z. Fu, and W. Britton at Boston Children’s Hospital for critical discussions. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

ACCORD The Action to Control Cardiovascular Risk in Diabetes Acox1 Acyl-CoA oxidase 1 AMD Age-related macular degeneration DR Diabetic retinopathy Fabp4 Fatty acid binding protein 4 VEGF-A Vascular endothelial growth factor A FGF21 Fibroblast growth factor 21 FIELD The Fenoﬁbrate Intervention and Event Lowering in Diabetes NV Neovascular tufts OIR Oxygen-induced retinopathy PPARα Peroxisome proliferator-activated receptor alpha RPE Retinal pigment epithelial SPPARMα Selective PPARα modulator VO Vaso-obliteration Abstract

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