Ryosuke Motohashi 1, Hidetaka Noma 1*, Kanako Yasuda 1, Osamu Kotake 1, Hiroshi Goto 2 and Masahiko Shimura 1

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1 Motohashi et al. Jounal of Inflammation (2018) 15:26 RESEARCH Open Access Dynamics of soluble vascula endothelial gowth facto eceptos and thei ligands in aqueous humou duing anibizumab fo age-elated macula degeneation Ryosuke Motohashi 1, Hidetaka Noma 1*, Kanako Yasuda 1, Osamu Kotake 1, Hioshi Goto 2 and Masahiko Shimua 1 Abstact Backgound: Intaviteal anibizumab injection (IRI) is effective fo patients with exudative age-elated macula degeneation (AMD) and deceases intaocula levels of vascula endothelial gowth facto (VEGF), but VEGF ecepto intaocula dynamics afte IRI ae unclea. Theefoe, we evaluated changes in the aqueous humo levels of soluble vascula endothelial gowth facto ecepto (svegfr)-1, svegfr-2, and thei ligands fo these eceptos (VEGF) patients with AMD eceiving IRI. Methods: The subjects wee 24 patients with AMD (24 eyes) who eceived 3 doses of IRI at monthly intevals. Aqueous humo samples wee obtained when each IRI dose was given (visits 0, 1, and 2 at 4-week intevals). Then the suspension aay method was employed to measue svegfr-1, svegfr-2, VEGF, and placental gowth facto (PlGF) in aqueous humo samples fom the 24 AMD patients and 13 cataact patients (as contols). Best coected visual acuity (BCVA; logmar) chat and cental macula thickness (CMT; optical coheence tomogaphy) wee also assessed ove time. Results: At baseline, the aqueous humo levels of svegfr-1, svegfr-2, VEGF, and PlGF wee significantly highe in the AMD goup than in the contol goup. Thee was a significant coelation between VEGF and PlGF o between svegfr-1 and svegfr-2. BCVA and CMT both impoved significantly afte IRI, and the aqueous humo levels of VEGF, PlGF, and svegfr-1 also deceased significantly. Conclusions: VEGFRs may be involved in the pathogenesis of AMD. IRI impoves clinical paametes in AMD patients by suppessing intaocula levels of VEGF, PlGF, and svegfr-1. Backgound Age-elated macula degeneation (AMD) is an impotant cause of visual loss [1]. In neovascula AMD, chooidal neovasculaization (CNV) leads to sub-etinal/ inta-etinal macula edema, hemohage, and fibosis, esulting in visual impaiment. Development of agents tageting vascula endothelial gowth facto (VEGF) has advanced consideably since an impotant ole of VEGF in the pathogenesis of CNV was epoted [2]. Fou anti-vegf agents ae available fo AMD, including an aptame of VEGF (pegaptanib), two anti-vegf * Coespondence: noma-hide@umin.ac.jp 1 Depatment of Ophthalmology, Hachioji Medical Cente, Tokyo Medical Univesity, 1163, Tatemachi, Hachioji, Tokyo , Japan Full list of autho infomation is available at the end of the aticle antibodies (bevacizumab and anibizumab) and a VEGF tapping agent (aflibecept) [3 5]. Howeve, ecuence of CNV afte teatment o esistance to these agents is not uncommon; suggesting a ole of othe factos in CNV along with VEGF. Viteous fluid levels of soluble VEGF ecepto (svegfr)-1 and svegfr-2 ae epoted to be elevated in ischemic viteoetinal diseases [6], and these eceptos may have a ole in the pathogenesis of macula edema [7]. Inteactions between the VEGFRs and thei ligands of these eceptos (VEGF) may influence pogession of AMD. While intaviteal anibizumab injection (IRI) has been epoted to educe intaocula VEGF [8]. intaocula dynamics of the VEGF eceptos afte IRI emain unclea. The Autho(s) Open Access This aticle is distibuted unde the tems of the Ceative Commons Attibution 4.0 Intenational License ( which pemits unesticted use, distibution, and epoduction in any medium, povided you give appopiate cedit to the oiginal autho(s) and the souce, povide a link to the Ceative Commons license, and indicate if changes wee made. The Ceative Commons Public Domain Dedication waive ( applies to the data made available in this aticle, unless othewise stated.

2 Motohashi et al. Jounal of Inflammation (2018) 15:26 Page 2 of 6 Accodingly, a pospective study was pefomed in which the concentations of VEGFR-1, VEGFR-2, and thei ligands (VEGF and placental gowth facto (PlGF)) wee measued in aqueous humos samples obtained fom patients with wet AMD at initiation of IRI, and changes of these factos wee evaluated duing the fist couse of IRI teatment. Methods Subjects This study was caied out at the Depatment of Ophthalmology of Tokyo Medical Univesity Hachioji Medical Cente (Tokyo, Japan) and was appoved by the Ethics Committee of the Cente. Study pocedues confomed to the Declaation of Helsinki and all patients gave witten infomed consent to enollment. Twenty-fou AMD patients (24 eyes) who wee scheduled to eceive thee doses of IRI (Lucentis; 0.5 mg/0.05 ml; Genentech, Inc., South San Fancisco, CA) at monthly intevals wee studied fom July 2013 to Mach None of the patients had peviously eceived teatment fo AMD. Aqueous humo samples wee havested just befoe IRI. Contol aqueous humo samples wee collected duing cataact sugey fom 13 patients (13 eyes) without othe ocula diseases. Best-coected visual acuity (BCVA) was deteminedbyusingalogaithmoftheminimumangleof esolution (LogMAR) chat. Patients with a histoy of etinal disease othe than AMD, glaucoma, uveitis, diabetes, ubeosis iidis, ocula infection, pio lase photocoagulation, o pevious intaocula sugey wee excluded fom the study. Fundus findings In all patients, funduscopy evealed elevated oange-ed lesions and/o chaacteistic polypoidal lesions wee found by indocyanine geen angiogaphy (IA). Eleven patients had typical wet-type AMD and 13 patients had polypoidal chooidal vasculopathy (PCV) based on the esults of fundus examination and IA, but none of them showed etinal angiomatous polifeation (RAP). Optical coheence tomogaphy (OCT) was pefomed within 1 week befoe IRI, employing a spectal-domain appaatus (Spectalis, Heidelbeg Engineeing, Heidelbeg, Gemany). The cental macula thickness (CMT) was measued with OCT softwae by two etinal specialists who wee blinded to infomation on the subjects. The seveity of macula edema was detemined fom the CMT. Pefomance of IRI Each patient eceived thee doses of IRI at 4-week intevals. Just befoe each injection, 0.1 ml of aqueous humo was havested by anteio chambe limbal paacentesis using a 30-gauge needle and an insulin syinge. Then IRI was administeed though the pas plana at 3.5 mm fom the limbus. Immediately afte collection, the aqueous humo samples wee tansfeed to steile plastic tube and stoed at 80 C until analysis. Measuement of cytokines and gowth factos Samples wee analyzed by using a suspension aay (xmap; Luminex Cop. Austin, TX) [9, 10] and captue bead kits (Beadlyte; Upstate Biotechnology, Lake Placid, NY) fo svegfr-1, svegfr-2, VEGF, and PlGF accoding to the manufactue s instuctions. Standad cuves wee geneated in duplicate fo each cytokine by using the cytokine efeence set supplied with the kit. To avoid between-un impecision, we measued the samples fom all patients in a single un. Cytokine levels wee within the assay detection anges (minimum detectable concentation was 1.59 pg/ml fo svegfr-1, pg/ml fo svegfr-2, 0.64 pg/ml fo VEGF, and 0.37 pg/ml fo PlGF). Statistical analysis Analyses wee pefomed with SAS System 9.4 softwae (SAS Institute Inc., Cay, Noth Caolina, USA). Student s t-test was employed to compae nomally distibuted unpaied continuous vaiables between the two goups, while the Mann-Whitney U test was used fo othe vaiables with a skewed distibution. Diffeences between the median aqueous levels wee assessed with the Wilcoxon single-ank test. To examine elationships among the vaiables, Speaman s ank-ode coelation coefficients o Peason s coelation coefficients wee calculated. Dunnet multiple compaison method was also applied. Two-tailed p values of less than 0.05 wee consideed to indicate statistical significance. Results The AMD goup (18 men and 6 women) was 76.0 ± 10.6 yeas old (mean ± SD) and the contol goup (7 men and 6 women) was 73.1 ± 5.9 yeas old. Thee wee no significant diffeences of age (p =0.369)ogende(p =0.189) between two goups. At the baseline examination (visit 0: just befoe the fist IRI dose), the mean BCVA was 0.39 ± 0.34 in the AMD goup and mean CMT was 401 ± 216 μm. As shown in Table 1, the aqueous humo levels of VEGF, PlGF, svegfr-1, and svegfr-2 wee significantly highe in the AMD goup than in the contol goup (in descending ode). In the AMD goup, thee was a significant coelation between the levels of svegfr-1 and svegfr-2, as well as between VEGF and PlGF levels (Table 2), but thee wee no coelations between svegfr-1 and VEGF o PlGF (Table 2) o between svegfr-2 and VEGF o PlGF (Table 2).

3 Motohashi et al. Jounal of Inflammation (2018) 15:26 Page 3 of 6 Table 1 Aqueous Humo Factos in the Contol and AMD Goups Paametes AMD Goup (N = 24) Contol Goup (N = 13) VEGF (pg/ml) 24.0 [ ] 0.64 [ ] PlGF (pg/ml) 1.59 [ ] 0.37 [ ] svegfr-1 (pg/ml) 466 [ ] 47.1 [ ] svegfr-2 (pg/ml) 405 [ ] 104 [ ] AMD age-elated macula degeneation, VEGF vascula endothelial gowth facto, PlGF plancental gowth facto, svegfr soluble vascula endothelial gowth facto ecepto In the AMD goup, mean BCVA showed significantly impvement fom log MAR 0.39 ± 0.34 befoe IRI to log MAR 0.28 ± 0.33 afte 1 month and log MAR 0.25 ± 0.36 afte 2 months (Fig. 1a), while mean CMT deceased significantly fom 401 ± 216 μm to 296 ± 199 μm afte 1 month and 267 ± 161 μm afte 2 months (Fig. 1b). In the AMD goup, the aqueous level of VEGF was significantly lowe at 1 and at 2 months afte stating IRI compaed with baseline (Fig. 2a). The aqueous level of PlGF was significantly lowe at 1 month and 2 months elative to baseline (Fig. 2b). The aqueous level of svegfr-1 was also significantly lowe at 1 and at 2 months elative to baseline (Fig. 3a). Howeve, thee was no significant change of the aqueous svegfr-2 level between baseline and 1 o 2 months (Fig. 3b). Discussion The main new findings wee that aqueous levels of both svegfr-1 and svegfr-2 wee significantly highe in the AMD goup than the contol goup, with a significant coelation between svegfr-1 and svegfr-2 levels, suggesting that expession of these eceptos shows concomitant upegulation in AMD. We ecently epoted [9, 10] that enhanced expession of both tansmembane VEGFR and svegfr is involved in egulating angiogenesis and vascula pemeability based on the coelation between thei expession in human umbilical vein endothelial cells stimulated with phobol 12-myistate 13-acetate o basic fiboblast gowth facto [11]. VEGF signaling may be tightly egulated by Table 2 Coelation Matix Fo Aqueous Humo Factos Vaiable VEGF PlGF svegfr-1 svegfr-2 VEGF 0.86 < PlGF svegfr VEGF vascula endothelial gowth facto, PlGF plancental gowth facto, svegfr soluble vascula endothelial gowth facto ecepto, coelation coefficient VEGFR-1 and VEGFR-2 [12]. VEGF binds to these eceptos on vascula endothelial cells, and both eceptos have tyosine kinase activity [13]. VEGFR-1 is also expessed by monocytes/macophages [14] and pomotes VEGF-mediated ecuitment of these cells to sites of angiogenesis and inflammation [14, 15]. On the othe hand, VEGFR-2 is only expessed by endothelial cells, and VEGF signaling via its membane-bound isofom influences physiological vascula pemeability and angiogenesis [12, 16]. These actions of the VEGF eceptos indicate the potential fo an impotant ole in the pathogenesis of AMD. Both svegfrs wee inceased in ou AMD patients, suggesting that these eceptos may be involved in development of CNV, and that the incease of svegfrs may be seconday to upegulation of tansmembane VEGFRs. We also demonstated that the aqueous humo level of VEGF was significantly suppessed in AMD patients by anti-vegf theapy, possibly because anibizumab binds and neutalize intaocula VEGF. These findings ae also in ageement with pevious epots [8]. IRI also significantly educed the aqueous humo level of PlGF, which was an unexpected finding VEGF and PlGF exist as dimes in the eye, being eithe homodimes (VEGF/VEGF and PlGF/PlGF) o heteodimes (VEGF/PlGF) [17, 18]. Theefoe, it is possible that the aqueous humo level of PlGF was educed significantly because anibizumab bound to and neutalized intaocula VEGF/PlGF heteodimes. Because VEGF acts as a chemoattactant fo monocytes afte binding to VEGFR-1 and pomotes inflammation [19], it is also possible that inflammation was suppessed by anibizumab binding and neutalizing VEGF. Accodingly, IRI may have educed inflammation in AMD patients to indiectly down-egulate PlGF expession. Inteestingly, we noted a significant decease in the aqueous level of svegfr-1 afte IRI teatment. VEGFR-1 is expessed by monocytes/macophages [14], and signaling via this ecepto pomotes VEGF-mediated ecuitment of these cells to sites of inflammation [14, 20]. It has been epoted that svegfr-1 has a po-inflammatoy effect [14, 21], suggesting that elevation of its expession may aggavate inflammation. This idea is suppoted by the epot that svegfr-1

4 Motohashi et al. Jounal of Inflammation (2018) 15:26 Page 4 of 6 Fig. 1 Pofile of the mean BCVA and mean CMT afte IRI in patients with AMD. a Mean BCVA impoved significantly fom log MAR 0.39 ± 0.34 to log MAR 0.28 ± 0.33 at 1 month (p = 0.002) and to log MAR 0.25 ± 0.36 at 2 months (p =0.004).b Mean CMT deceased significantly fom 401 ± 216 μm to 296 ± 199 μm at 1 month (p < 0.001) and to 267 ± 161 μm at 2 months (P <0.001) expession is coelated with the C-eactive potein level and the leukocyte count [22], which shows a possible link between svegfr-1 and inflammation. Chonic inflammationmayhaveaoleinthepathogenesisofamd[23], and VEGF is known to be a chemoattactant fo monocytes in pimates [24]. Thus, inflammation may be suppessed by anibizumab binding and neutalizing VEGF and this suppession of inflammation by IRI could be associated with down-egulation of svegfr-1 in AMD eyes. On the othe hand, thee was no significant decease of svegfr-2 expession duing IRI, suggesting that its effects ae associated with down-egulation of VEGFR-1 athe than VEGFR-2. Because VEGFR-2 signaling mainly influences vascula pemeability and angiogenesis [12], the svegfr-2 level might not decease significantly when IRI suppesses inflammation. Howeve, this study had a small sample size and futhe investigation will be equied to claify the kinetics of svegfrs in AMD patients eceiving IRI. Accoding to ou findings, impovement of BCVA and CMT occued duing IRI along with eduction of the aqueous humo levels of VEGF, PlGF, and svegfr-1. Theefoe, IRI may achieve functional and mophological impovement in AMD patients by suppessing intaocula poduction of VEGF, PlGF, and svegfr-1. Fig. 2 Changes in the aqueous humo level of VEGF o PlGF afte IRI in patients with AMD. a The aqueous humo level of VEGF showed a significant decease at 1 month (0.64 pg/ml [ ]) and at 2 months (0.64 pg/ml [ ]) compaed with baseline (24.0 pg/ml [ ]) (P < and P < 0.001, espectively). b The aqueous humo level of PlGF showed a significant decease at 1 month (1.27 pg/ml [ ]) and at 2 months (1.17 pg/ml [ ]) compaed with baseline (1.59 pg/ml [ ]) (P = and P < 0.001, espectively)

5 Motohashi et al. Jounal of Inflammation (2018) 15:26 Page 5 of 6 Fig. 3 Changes in the aqueous humo level of svegfr-1 o svegfr-1 afte IRI in patients with AMD. a Aqueous humo levels of svegfr-1 showed a significant decease at 1 month (268 pg/ml [ ]) and at 2 months (223 pg/ml [ ]) compaed with baseline (466 pg/ml [ ]) (P = and P < 0.001, espectively). b Thee was no significant diffeence in the aqueous humo level of svegfr-2 between baseline (405 pg/ml [ ]) and 1 month (372 pg/ml [ ]) o 2 months (276 pg/ml [ ]) (P = and P = 0.303, espectively) Conclusions In conclusion, the levels of svegfr-1, svegfr-2, VEGF, and PlGF in the aqueous humo wee significantly highe in patients with AMD than in contols. Aqueous levels of svegfr-1 and svegfr-2 showed a significant coelation with each othe, as did the levels of VEGF and PlGF. IRI significantly impoved both BCVA and CMT, while aqueous levels of VEGF, PlGF, and svegfr-1 deceased significantly. These findings suggest that VEGFRs may have a ole in AMD and that suppession of intaocula VEGF, PlGF, and svegfr-1 poduction by IRI may impove clinical paametes. Abbeviations AMD: Age-elated macula degeneation; BCVA: Best-coected visual acuity; CMT: Cental macula thickness; CNV: Chooidal neovasculaization; IA: Indocyanine geen angiogaphy; IRI: Intaviteal anibizumab injection; logmar: Logaithm of minimal angle of esolution; OCT: Optical coheence tomogaphy; PCV: Polypoidal chooidal vasculopathy; PlGF: Placental gowth facto; RAP: Retinal angiomatous polifeation; svegfr: Soluble VEGF ecepto; VEGF: Vascula endothelial gowth facto Acknowledgements We thank Katsunoi Shimada (Depatment of Biostatistics, STATZ Copoation, Tokyo) fo assistance with the statistical analysis. Funding None. Availability of data and mateials Data will not be shaed because the authos ae pefoming othe analyses, which have not yet been published. Authos contibutions HN, and MS wee involved in the design and conduct of the study. Collection and management of the data wee done by RM, HN, KY, OK, and MS, while analysis and intepetation of the data wee pefomed by HN, HG, and MS. Pepaation of the fist daft of the manuscipt was done by R.M., and eview and appoval of the manuscipt was pefomed by HN, HG, and MS. All authos ead and appoved the final manuscipt. Ethics appoval and consent to paticipate This study was conducted at the Depatment of Ophthalmology of Tokyo Medical Univesity and appoval was obtained fom the Univesity Ethics Committee. The pocedues of the study confomed to the tenets of the Declaation of Helsinki and all patients gave witten infomed consent befoe enollment. Consent fo publication Witten infomed consent fo publication of clinical images was obtained fom the individual patients. The details of consent to publication wee included in the consent fom fo paticipation in this study. Competing inteests The authos declae that they have no competing inteests. Publishe s Note Spinge Natue emains neutal with egad to juisdictional claims in published maps and institutional affiliations. Autho details 1 Depatment of Ophthalmology, Hachioji Medical Cente, Tokyo Medical Univesity, 1163, Tatemachi, Hachioji, Tokyo , Japan. 2 Depatment of Ophthalmology, Tokyo Medical Univesity, Tokyo, Japan. Received: 9 Novembe 2018 Accepted: 22 Novembe 2018 Refeences 1. Jage RD, Miele WF, Mille JW. Age-elated macula degeneation. N Engl J Med. 2008;2: Rosenfeld PJ, Bown DM, Heie JS, Boye DS, Kaise PK, Chung CY, Kim RY. Ranibizumab fo neovascula age-elated macula degeneation. N Engl J Med. 2006;355: Cameliet P, Moons L, Luttun A, Vincenti V, Compenolle V, De Mol M, et al. Synegism between vascula endothelial gowth facto and placental gowth facto contibutes to angiogenesis and plasma extavasation in pathological conditions. Nat Med. 2001;7: Holash J, Davis S, Papadopoulos N, Coll SD, Ho L, Russell M, et al. VEGF- Tap: a VEGF blocke with potent antitumo effects. Poc Natl Acad Sci U S A. 2002;99: Heie JS, Antoszyk AN, Pavan PR, Leff SR, Rosenfeld PJ, Ciulla TA, et al. Ranibizumab fo teatment of neovascula age-elated macula degeneation: a phase I/II multicente, contolled, multidose study. Ophthalmology. 2006;113(633):e1 4.

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