Precise staging of beetle horn formation in Trypoxylus dichotomus reveals the pleiotropic roles of doublesex depending on the spatiotemporal developmental contexts.

Many scarab beetles have sexually dimorphic exaggerated horns that are an evolutionary novelty. Since the shape, number, size, and location of horns are highly diverged within Scarabaeidae, beetle horns are an attractive model for studying the evolution of sexually dimorphic and novel traits. In beetles including the Japanese rhinoceros beetle Trypoxylus dichotomus, the sex differentiation gene doublesex (dsx) plays a crucial role in sexually dimorphic horn formation during larval-pupal development. However, knowledge of when and how dsx drives the gene regulatory network (GRN) for horn formation to form sexually dimorphic horns during development remains elusive. To address this issue, we identified a Trypoxylus-ortholog of the sex determination gene, transformer (tra), that regulates sex-specific splicing of the dsx pre-mRNA, and whose loss of function results in sex transformation. By knocking down tra function at multiple developmental timepoints during larval-pupal development, we estimated the onset when the sex-specific GRN for horn formation is driven. In addition, we also revealed that dsx regulates different aspects of morphogenetic activities during the prepupal and pupal developmental stages to form appropriate morphologies of pupal head and thoracic horn primordia as well as those of adult horns. Based on these findings, we discuss the evolutionary developmental background of sexually dimorphic trait growth in horned beetles.

Rhinoceros beetle horn development reveals deep parallels with dung beetles.

Beetle horns are attractive models for studying the evolution of novel traits, as they display diverse shapes, sizes, and numbers among closely related species within the family Scarabaeidae. Horns radiated prolifically and independently in two distant subfamilies of scarabs, the dung beetles (Scarabaeinae), and the rhinoceros beetles (Dynastinae). However, current knowledge of the mechanisms underlying horn diversification remains limited to a single genus of dung beetles, Onthophagus. Here we unveil 11 horn formation genes in a rhinoceros beetle, Trypoxylus dichotomus. These 11 genes are mostly categorized as larval head- and appendage-patterning genes that also are involved in Onthophagus horn formation, suggesting the same suite of genes was recruited in each lineage during horn evolution. Although our RNAi analyses reveal interesting differences in the functions of a few of these genes, the overwhelming conclusion is that both head and thoracic horns develop similarly in Trypoxylus and Onthophagus, originating in the same developmental regions and deploying similar portions of appendage patterning networks during their growth. Our findings highlight deep parallels in the development of rhinoceros and dung beetle horns, suggesting either that both horn types arose in the common ancestor of all scarabs, a surprising reconstruction of horn evolution that would mean the majority of scarab species (~35,000) actively repress horn growth, or that parallel origins of these extravagant structures resulted from repeated co-option of the same underlying developmental processes.

Six-month results of intravitreal ranibizumab for macular edema after branch retinal vein occlusion in a single-center prospective study: visual outcomes and microaneurysm formation.

PURPOSE: The aim of this study is to report the 6-month results after one intravitreal ranibizumab (IVR) injection followed by pro re nata dosing for macular edema (ME) after branch retinal vein occlusion. PATIENTS AND METHODS: The inclusion criteria included a minimal patient age of 18 years, 20 letters or more best-corrected visual acuity (BCVA) (Early Treatment Diabetic Retinopathy Study [ETDRS] score, 77 letters or less), and central retinal thickness (CRT) of 250 microns or more. The primary outcome measure was the mean BCVA change from baseline at month 6; the secondary outcomes were mean changes in CRT, residual ME, and microaneurysm formation. RESULTS: Twenty patients were enrolled from March 2014 through October 2016 at Nagoya City University Hospital. The baseline mean ETDRS letters and CRT were 63.1 and 500 microns, respectively; mean time from symptom onset to initial therapy was 1.80 months; and mean ETDRS gain and CRT reduction were 15.2 letters and 230 microns, respectively. The percentages of patients with Snellen equivalent BCVAs of 20/40 (70 ETDRS letters) or better and 20/20 (85 ETDRS letters) were 90% and 15%, respectively. Residual ME and microaneurysms were observed in 85% and 35% of patients. Microaneurysm formation was associated with delayed initial therapy. CONCLUSION: Prompt initiation of IVR injection provided a better visual prognosis at month 6 and suppressed the microaneurysm formation.

Tissue Plasminogen Activator as an Antiangiogenic Agent in Experimental Laser-Induced Choroidal Neovascularization in Mice.

PURPOSE: We investigate the antiangiogenic efficacy of tissue plasminogen activator (tPA) on experimental laser-induced choroidal neovascularization (CNV) in mice. METHODS: After CNV was induced by laser photocoagulation in 92 C57BL/6J wild-type mice, tPA (4 or 40 international units [IU]/µl) or PBS was injected intravitreally immediately after laser injury. Fluorescein angiography was performed on day 7 to grade CNV leakage. The CNV volume was measured by confocal microscopy in eyes enucleated 7 days after laser injury. Immunohistochemical studies were performed 3 days after laser injury to evaluate fibrin/fibrinogen and CD31 expression. The possible adverse effects of tPA were assessed by electroretinography (ERG) and histology on day 7. RESULTS: Intravitreal administration of tPA significantly suppressed CNV leakage and CNV volume in a dose-dependent manner (P < 0.01). Intravitreal injection of tPA suppressed fibrin/fibrinogen and CD31 expression in laser-induced lesions. Histologic examination and ERG showed no evidence of retinal toxicity in eyes injected with tPA. CONCLUSIONS: Intravitreal injection of tPA suppressed fibrin/fibrinogen expression and laser-induced CNV. The current results suggested that tPA may be a potential therapeutic adjuvant for treating CNV.

Characterization of evolutionary trend in squamate estrogen receptor sensitivity.

Steroid hormones are a key regulator of reproductive biology in vertebrates, and are largely regulated via nuclear receptor families. Estrogen signaling is regulated by two estrogen receptor (ER) subtypes alpha and beta in the nucleus. In order to understand the role of estrogen in vertebrates, these ER from various species have been isolated and were functionally analyzed using luciferase reporter gene assays. Interestingly, species difference in estrogen sensitivity has been noted in the past, and it was reported that snake ER displayed highest estrogen sensitivity. Here, we isolated additional ER from three lizards: chameleon (Bradypodion pumilum), skink (Plestiodon finitimus), and gecko (Gekko japonicus). We have performed functional characterization of these ERs using reporter gene assay system, and found high estrogen sensitivity in all three species. Furthermore, comparison with results from other tetrapod ER revealed a seemingly uniform gradual pattern of ligand sensitivity evolution. In silico 3D homology modeling of the ligand-binding domain revealed structural variation at three sites, helix 2, and juncture between helices 8 and 9, and caudal region of helix 10/11. Docking simulations indicated that predicted ligand-receptor interaction also correlated with the reporter assay results, and overall squamates displayed highest stabilized interactions. The assay system and homology modeling system provides tool for in-depth comparative analysis of estrogen function, and provides insight toward the evolution of ER among vertebrates.

Intravenous immune globulin suppresses angiogenesis in mice and humans.

Human intravenous immune globulin (IVIg), a purified IgG fraction composed of ~ 60% IgG1 and obtained from the pooled plasma of thousands of donors, is clinically used for a wide range of diseases. The biological actions of IVIg are incompletely understood and have been attributed both to the polyclonal antibodies therein and also to their IgG (IgG) Fc regions. Recently, we demonstrated that multiple therapeutic human IgG1 antibodies suppress angiogenesis in a target-independent manner via FcγRI, a high-affinity receptor for IgG1. Here we show that IVIg possesses similar anti-angiogenic activity and inhibited blood vessel growth in five different mouse models of prevalent human diseases, namely, neovascular age-related macular degeneration, corneal neovascularization, colorectal cancer, fibrosarcoma and peripheral arterial ischemic disease. Angioinhibition was mediated by the Fc region of IVIg, required FcγRI and had similar potency in transgenic mice expressing human FcγRs. Finally, IVIg therapy administered to humans for the treatment of inflammatory or autoimmune diseases reduced kidney and muscle blood vessel densities. These data place IVIg, an agent approved by the US Food and Drug Administration, as a novel angioinhibitory drug in doses that are currently administered in the clinical setting. In addition, they raise the possibility of an unintended effect of IVIg on blood vessels.

Human IgG1 antibodies suppress angiogenesis in a target-independent manner.

Aberrant angiogenesis is implicated in diseases affecting nearly 10% of the world's population. The most widely used anti-angiogenic drug is bevacizumab, a humanized IgG1 monoclonal antibody that targets human VEGFA. Although bevacizumab does not recognize mouse Vegfa, it inhibits angiogenesis in mice. Here we show bevacizumab suppressed angiogenesis in three mouse models not via Vegfa blockade but rather Fc-mediated signaling through FcγRI (CD64) and c-Cbl, impairing macrophage migration. Other approved humanized or human IgG1 antibodies without mouse targets (adalimumab, alemtuzumab, ofatumumab, omalizumab, palivizumab and tocilizumab), mouse IgG2a, and overexpression of human IgG1-Fc or mouse IgG2a-Fc, also inhibited angiogenesis in wild-type and FcγR humanized mice. This anti-angiogenic effect was abolished by Fcgr1 ablation or knockdown, Fc cleavage, IgG-Fc inhibition, disruption of Fc-FcγR interaction, or elimination of FcRγ-initated signaling. Furthermore, bevacizumab's Fc region potentiated its anti-angiogenic activity in humanized VEGFA mice. Finally, mice deficient in FcγRI exhibited increased developmental and pathological angiogenesis. These findings reveal an unexpected anti-angiogenic function for FcγRI and a potentially concerning off-target effect of hIgG1 therapies.

Evolution of estrogen receptors in ray-finned fish and their comparative responses to estrogenic substances.

In vertebrates, estrogens play fundamental roles in regulating reproductive activities through estrogen receptors (ESRs), and disruption of estrogen signaling is now of global concern for both wildlife and human health. To date, ESRs of only a limited number of species have been characterized. We investigated the functional diversity and molecular basis or ligand sensitivity of ESRs among ray-finned fish species (Actinopterygii), the most variable group within vertebrates. We cloned and characterized ESRs from several key species in the evolution of ray-finned fish including bichir (Polypteriformes, ESR1 and ESR2) at the basal lineage of ray-finned fish, and arowana (Osteoglossiformes, ESR1 and ESR2b) and eel (Anguilliformes, ESR1, ESR2a and ESR2b) both belonging to ancient early-branching lineages of teleosts, and suggest that ESR2a and ESR2b emerged through teleost-specific whole genome duplication, but an ESR1 paralogue has been lost in the early lineage of euteleost fish species. All cloned ESR isoforms showed similar responses to endogenous and synthetic steroidal estrogens, but they responded differently to non-steroidal estrogenic endocrine disrupting chemicals (EDCs) (e.g., ESR2a exhibits a weaker reporter activity compared with ESR2b). We show that variation in ligand sensitivity of ESRs can be attributed to phylogeny among species of different taxonomic groups in ray-finned fish. The molecular information provided contributes both to understanding of the comparative role of ESRs in the reproductive biology of fish and their comparative responses to EDCs.

Nucleoside Reverse Transcriptase Inhibitors Suppress Laser-Induced Choroidal Neovascularization in Mice.

PURPOSE: To evaluate the efficacy of nucleoside reverse transcriptase inhibitors (NRTIs) in the laser-induced mouse model of choroidal neovascularization (CNV). METHODS: We evaluated the NRTIs lamivudine (3TC), zidovudine (AZT), and abacavir (ABC) and the P2X7 antagonist A438079. Choroidal neovascularization was induced by laser injury in C57BL/6J wild-type, Nlrp3-/-, and P2rx7-/- mice, and CNV volume was measured after 7 days by confocal microscopy. Drugs were administered by intravitreous injection immediately after the laser injury. Vascular endothelial growth factor-A in RPE-choroid lysates was measured 3 days after laser injury by ELISA. HEK293 cells expressing human and mouse P2X7 were exposed to the selective P2X7 receptor agonist 2', 3'-(benzoyl-4-benzoyl)-ATP (Bz-ATP) with or without 3TC, and VEGF-A levels in media were measured by ELISA. RESULTS: Intravitreous injection of 3TC, AZT, and ABC significantly suppressed laser-induced CNV in C57BL/6J wild-type and Nlrp3-/- mice (P < 0.05) but not in P2rx7-/- mice. Intravitreous injection of A438079 also suppressed the laser-induced CNV (P < 0.05). The NRTIs 3TC, AZT, and ABC blocked VEGF-A levels in the RPE/choroid after laser injury in wild-type (P < 0.05) but not P2rx7-/- mice. Moreover, there was no additive effect of 3TC on CNV inhibition when coadministered with a neutralizing VEGF-A antibody. Stimulation of human and mouse P2X7-expressing HEK293 cells with Bz-ATP increased VEGF secretion (P < 0.001), which was abrogated by 3TC (P < 0.001). Stimulation of primary human RPE cells with Bz-ATP increased VEGFA and IL6 mRNA levels, which were abrogated by 3TC. CONCLUSIONS: Multiple clinically relevant NRTIs suppressed laser-induced CNV and downregulated VEGF-A, via P2X7.

Environmental chemicals active as human antiandrogens do not activate a stickleback androgen receptor but enhance a feminising effect of oestrogen in roach.

Sexual disruption is reported in wild fish populations living in freshwaters receiving discharges of wastewater treatment works (WwTW) effluents and is associated primarily with the feminisation of males by exposure to oestrogenic chemicals. Antiandrogens could also contribute to the feminisation of male fish, but there are far less data supporting this hypothesis and almost nothing is known for the effects of oestrogens in combination with antiandrogens in fish. We conducted a series of in vivo exposures in two fish species to investigate the potency on reproductive-relevant endpoints of the antiandrogenic antimicrobials triclosan (TCS), chlorophene (CP) and dichlorophene (DCP) and the resin, abietic acid (AbA), all found widely in WwTW effluents. We also undertook exposures with a mixture of antiandrogens and a mixture of antiandrogens in combination with the oestrogen 17α-ethinyloestradiol (EE2). In stickleback (Gasterosteus aculeatus), DCP showed a tendency to reduce spiggin induction in females androgenised by dihydrotestosterone (DHT), but these findings were not conclusive. In roach (Rutilus rutilus), exposures to DCP (178 days), or a mixture of TCS, CP and AbA (185 days), or to the model antiandrogen flutamide (FL, 178 days) had no effect on gonadal sex ratio or on the development of the reproductive ducts. Exposure to EE2 (1.5ng/L, 185 days) induced feminisation of the ducts in 17% of the males and in the mixture of antiandrogens (TCS, CP, AbA) in combination with EE2, almost all (96%) of the males had a feminised reproductive ducts. In stickleback androgen receptor (ARα and ARß) transactivation assays, the model antiandrogens, FL and procymidone inhibited 11-ketotestosterone (11-KT) induced receptor activation, but none of the human antiandrogens, TCS, CP, DCP and AbA had an effect. These data indicate that antimicrobial antiandrogens in combination can contribute to the feminisation process in exposed males, but they do not appear to act through the androgen receptor in fish.

Understanding the molecular basis for differences in responses of fish estrogen receptor subtypes to environmental estrogens.

Exposure to endocrine disrupting chemicals (EDCs) can elicit adverse effects on development, sexual differentiation, and reproduction in fish. Teleost species exhibit at least three subtypes of estrogen receptor (ESR), ESR1, ESR2a, and ESR2b; thus, estrogenic signaling pathways are complex. We applied in vitro reporter gene assays for ESRs in five fish species to investigate the ESR subtype-specificity for better understanding the signaling pathway of estrogenic EDCs. Responses to bisphenol A, 4-nonylphenol, and o,p'-DDT varied among ESR subtypes, and the response pattern of ESRs was basically common among the different fish species. Using a computational in silico docking model and through assays quantifying transactivation of the LBD (using GAL-LBD fusion proteins and chimera proteins for the ESR2s), we found that the LBD of the different ESR subtypes generally plays a key role in conferring responsiveness of the ESR subtypes to EDCs. These results also indicate that responses of ESR2s to EDCs cannot necessarily be predicted from the LBD sequence alone, and an additional region is required for full transactivation of these receptors. Our data thus provide advancing understanding on receptor functioning for both basic and applied research.

Characterization of Oryzias latipes glucocorticoid receptors and their unique response to progestins.

Various receptor bioassays, including estrogens, androgens and thyroid hormones, have been developed and applied successfully for assessing hormone function in a wide range of animal species, including fish. In fish, corticosteroids play a pivotal role in physiology as they do in mammals, but far less is known about the corticosteroid receptor system in fish compared with in mammals. Here we established a transient transactivation assay using the Japanese medaka, Oryzias latipes, glucocorticoid receptors (olGRs) and mineralocorticoid receptor to analyse their functional properties in a fish. We found that olGR2 was highly responsive to glucocorticoids, similar to the human GR, whereas the olGR1 subtype was minimally responsive. Thus, olGR2 most likely mediates glucocorticoid signaling in medaka. We further tested crosstalk between GRs and other steroid hormones, and found that progestins could activate or inactivate olGR2-mediating transcription, depending on the presence or absence of cortisol. The transactivation assays developed for medaka GRs provide tools to gain useful insights into corticosteroid signaling in fish and for in vitro screening of environmental substances activating GRs.

Nucleoside reverse transcriptase inhibitors possess intrinsic anti-inflammatory activity.

Nucleoside reverse transcriptase inhibitors (NRTIs) are mainstay therapeutics for HIV that block retrovirus replication. Alu (an endogenous retroelement that also requires reverse transcriptase for its life cycle)-derived RNAs activate P2X7 and the NLRP3 inflammasome to cause cell death of the retinal pigment epithelium in geographic atrophy, a type of age-related macular degeneration. We found that NRTIs inhibit P2X7-mediated NLRP3 inflammasome activation independent of reverse transcriptase inhibition. Multiple approved and clinically relevant NRTIs prevented caspase-1 activation, the effector of the NLRP3 inflammasome, induced by Alu RNA. NRTIs were efficacious in mouse models of geographic atrophy, choroidal neovascularization, graft-versus-host disease, and sterile liver inflammation. Our findings suggest that NRTIs are ripe for drug repurposing in P2X7-driven diseases.

DICER1/Alu RNA dysmetabolism induces Caspase-8-mediated cell death in age-related macular degeneration.

Geographic atrophy, an advanced form of age-related macular degeneration (AMD) characterized by death of the retinal pigmented epithelium (RPE), causes untreatable blindness in millions worldwide. The RPE of human eyes with geographic atrophy accumulates toxic Alu RNA in response to a deficit in the enzyme DICER1, which in turn leads to activation of the NLRP3 inflammasome and elaboration of IL-18. Despite these recent insights, it is still unclear how RPE cells die during the course of the disease. In this study, we implicate the involvement of Caspase-8 as a critical mediator of RPE degeneration. Here we show that DICER1 deficiency, Alu RNA accumulation, and IL-18 up-regulation lead to RPE cell death via activation of Caspase-8 through a Fas ligand-dependent mechanism. Coupled with our observation of increased Caspase-8 expression in the RPE of human eyes with geographic atrophy, our findings provide a rationale for targeting this apoptotic pathway in this disease.

Differing species responsiveness of estrogenic contaminants in fish is conferred by the ligand binding domain of the estrogen receptor.

Exposure to estrogenic endocrine disrupting chemicals (EDCs) induces a range of adverse effects, notably on reproduction and reproductive development. These responses are mediated via estrogen receptors (ERs). Different species of fish may show differences in their responsiveness to environmental estrogens but there is very limited understanding on the underlying mechanisms accounting for these differences. We used custom developed in vitro ERα reporter gene assays for nine fish species to analyze the ligand- and species-specificity for 12 environmental estrogens. Transcriptonal activities mediated by estradiol-17ß (E2) were similar to only a 3-fold difference in ERα sensitivity between species. Diethylstilbestrol was the most potent estrogen (∼ 10-fold that of E2) in transactivating the fish ERαs, whereas equilin was about 1 order of magnitude less potent in all species compared to E2. Responses of the different fish ERαs to weaker environmental estrogens varied, and for some considerably. Medaka, stickleback, bluegill and guppy showed higher sensitivities to nonylphenol, octylphenol, bisphenol A and the DDT-metabolites compared with cyprinid ERαs. Triclosan had little or no transactivation of the fish ERαs. By constructing ERα chimeras in which the AF-containing domains were swapped between various fish species with contrasting responsiveness and subsequent exposure to different environmental estrogens. Our in vitro data indicate that the LBD plays a significant role in accounting for ligand sensitivity of ERα in different species. The differences seen in responsiveness to different estrogenic chemicals between species indicate environmental risk assessment for estrogens cannot necessarily be predicted for all fish by simply examining receptor activation for a few model fish species.

Molecular impact of juvenile hormone agonists on neonatal Daphnia magna.

Daphnia magna has been used extensively to evaluate organism- and population-level responses to pollutants in acute toxicity and reproductive toxicity tests. We have previously reported that exposure to juvenile hormone (JH) agonists results in a reduction of reproductive function and production of male offspring in a cyclic parthenogenesis, D. magna. Recent advances in molecular techniques have provided tools to understand better the responses to pollutants in aquatic organisms, including D. magna. DNA microarray was used to evaluate gene expression profiles of neonatal daphnids exposed to JH agonists: methoprene (125, 250 and 500 ppb), fenoxycarb (0.5, 1 and 2 ppb) and epofenonane (50, 100 and 200 ppb). Exposure to these JH analogs resulted in chemical-specific patterns of gene expression. The heat map analyses based on hierarchical clustering revealed a similar pattern between treatments with a high dose of methoprene and with epofenonane. In contrast, treatment with low to middle doses of methoprene resulted in similar profiles to fenoxycarb treatments. Hemoglobin and JH epoxide hydrolase genes were clustered as JH-responsive genes. These data suggest that fenoxycarb has high activity as a JH agonist, methoprene shows high toxicity and epofenonane works through a different mechanism compared with other JH analogs, agreeing with data of previously reported toxicity tests. In conclusion, D. magna DNA microarray is useful for the classification of JH analogs and identification of JH-responsive genes.

Molecular cloning of doublesex genes of four cladocera (water flea) species.

BACKGROUND: The gene doublesex (dsx) is known as a key factor regulating genetic sex determination in many organisms. We previously identified two dsx genes (DapmaDsx1 and DapmaDsx2) from a freshwater branchiopod crustacean, Daphnia magna, which are expressed in males but not in females. D. magna produces males by parthenogenesis in response to environmental cues (environmental sex determination) and we showed that DapmaDsx1 expression during embryonic stages is responsible for the male trait development. The D. magna dsx genes are thought to have arisen by a cladoceran-specific duplication; therefore, to investigate evolutionary conservation of sex specific expression of dsx genes and to further assess their functions in the environmental sex determination, we searched for dsx homologs in four closely related cladoceran species. RESULTS: We identified homologs of both dsx genes from, D. pulex, D. galeata, and Ceriodaphnia dubia, yet only a single dsx gene was found from Moina macrocopa. The deduced amino acid sequences of all 9 dsx homologs contained the DM and oligomerization domains, which are characteristic for all arthropod DSX family members. Molecular phylogenetic analysis suggested that the dsx gene duplication likely occurred prior to the divergence of these cladoceran species, because that of the giant tiger prawn Penaeus monodon is rooted ancestrally to both DSX1 and DSX2 of cladocerans. Therefore, this result also suggested that M. macrocopa lost dsx2 gene secondarily. Furthermore, all dsx genes identified in this study showed male-biased expression levels, yet only half of the putative 5' upstream regulatory elements are preserved in D. magna and D. pulex. CONCLUSIONS: The all dsx genes of five cladoceran species examined had similar amino acid structure containing highly conserved DM and oligomerization domains, and exhibited sexually dimorphic expression patterns, suggesting that these genes may have similar functions for environmental sex determination in cladocerans.

Suppression of laser-induced choroidal neovascularization by a CCR3 antagonist.

PURPOSE: To evaluate the efficacy of a novel CCR3 antagonist for laser injury-induced choroidal neovascularization (CNV) in mice. METHODS: We evaluated YM-344031, a novel and selective small-molecule CCR3 antagonist. CNV was induced by laser injury in C57BL/6J mice, and its volume was measured after 7 days by confocal microscopy. Leakage from the CNV was also measured after 7 days by fluorescein angiography. The CCR3 antagonist was administered by gavage at 1 hour before and 1 day after the laser injury, or intravitreous injection immediately after the laser injury. After the laser injury, ELISA, Western blot analysis, and real-time RT-PCR for VEGF-A expression in the RPE/choroid, and immunohistochemistry for CCR3, CCL11, Ki67, and Rac1 was performed. RESULTS: Both oral administration and intravitreous injection of YM-344031 significantly suppressed the CNV volume (P < 0.0001 and P < 0.01, respectively). Pathologically significant leakage was significantly less common in YM-344031-injected mice (P < 0.0001). The mean VEGF protein level was significantly increased in vehicle-injected eyes after the laser injury (P < 0.05). Although the YM-344031-injected eyes did not show VEGF-A suppression after the laser injury, VEGF164 mRNA upregulation was significantly suppressed in YM-344031-injected mice (P < 0.05), and intravitreous injection of YM-344031 appeared to suppress CCR3, CCL11 (eotaxin), Ki67, and Rac1 expression after the laser injury. CONCLUSIONS: The present data suggest that the CCR3 antagonist YM-344031 can suppress CNV, via suppression of the upregulation of VEGF164 mRNA in VEGF isoform after the laser injury. Although our findings may warrant further investigation, YM-344031 may have potential as a new therapy for age-related macular degeneration.