Suppression and Regression of Choroidal Neovascularization in Mice by a Novel CCR2 Antagonist, INCB3344 Ping Xie, Motohiro Kamei*, Mihoko Suzuki, Nagakazu Matsumura, Kentaro Nishida, Susumu Sakimoto, Hirokazu Sakaguchi, Kohji Nishida Department of Ophthalmology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Abstract Purpose: To investigate the effect of an intravitreally administered CCR2 antagonist, INCB3344, on a mouse model of choroidal neovascularization (CNV). Methods: CNV was induced by laser photocoagulation on Day 0 in wild type mice. INCB3344 or vehicle was administered intravitreally immediately after laser application. On Day 14, CNV areas were measured on retinal pigment epithelium (RPE)- choroid flat mounts and histopathologic examination was performed on 7 mm-thick sections. Macrophage infiltration was evaluated by immunohistochemistry on RPE-choroid flat mounts and quantified by flow cytometry on Day 3. Expression of vascular endothelial growth factor (VEGF) protein in RPE-choroid tissue was examined by immunohistochemistry and ELISA, VEGF mRNA in sorted macrophages in RPE-choroid tissue was examine by real-time PCR and expression of phosphorylated extracellular signal-regulated kinase (p-ERK 1/2) in RPE-choroid tissue was measured by Western blot analysis on Day 3. We also evaluated the efficacy of intravitreal INCB3344 to spontaneous CNV detected in Cu, Zn-superoxide dismutase (SOD1) deficient mice. Changes in CNV size were assessed between pre- and 1week post-INCB3344 or vehicle administration in fundus photography and fluorescence angiography (FA). Results: The mean CNV area in INCB3344-treated mice decreased by 42.4% compared with the vehicle-treated control mice (p,0.001). INCB3344 treatment significantly inhibited macrophage infiltration into the laser-irradiated area (p,0.001), and suppressed the expression of VEGF protein (p = 0.012), VEGF mRNA in infiltrating macrophages (p,0.001) and the phosphorylation of ERK1/2 (p,0.001). The area of spontaneous CNV in Sod12/2 mice regressed by 70.35% in INCB3344- treated animals while no change was detected in vehicle-treated control mice (p,0.001). Conclusions: INCB3344 both inhibits newly forming CNV and regresses established CNV. Controlling inflammation by suppressing macrophage infiltration and angiogenic ability via the CCR-2/MCP-1 signal may be a useful therapeutic strategy for treating CNV associated with age-related macular degeneration. Citation: Xie P, Kamei M, Suzuki M, Matsumura N, Nishida K, et al. (2011) Suppression and Regression of Choroidal Neovascularization in Mice by a Novel CCR2 Antagonist, INCB3344. PLoS ONE 6(12): e28933. doi:10.1371/journal.pone.0028933 Editor: Paul Proost, University of Leuven, Rega Institute, Belgium Received August 9, 2011; Accepted November 17, 2011; Published December 19, 2011 Copyright: 2011 Xie et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a Grant-in-Aid for Scientific Research (#15591853) from the Ministry of Education, Science and Culture of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mkamei@ophthal.med.osaka-u.ac.jp Introduction Age-related macular degeneration (AMD) is the leading cause of legal blindness among elderly people in developed countries [1]. The majority of these patients with severe vision loss have retinal damage by choroidal neovascularization (CNV) [2], which is the hallmark of wet AMD and is defined as new blood vessels arising from choriocapillaris and extending into the sub-retinal pigment epithelium (sub-RPE) [3], sub-retinal space, or both. Although the pathogenesis of CNV is not completely understood, inflammatory processes, especially the infiltration of inflammatory cells, are recognized as an important mediator of CNV formation [4]. Macrophage accumulation in the CNV area and expression of angiogenic cytokines, such as vascular endothelial growth factor (VEGF) are involved in CNV formation [5–10]. Moreover, in mice depleted of macrophages, CNV is reduced and VEGF production is decreased [11], [12], which suggests a role for macrophages as producers and regulators of angiogenic factors in the pathogenesis of CNV. The chemokine receptor CCR2 and its ligand, monocyte chemoattractant protein-1 (MCP-1; also known as CCL2) represent a critical signaling pathway responsible for the recruitment of monocyte-macrophages [13–16]. MCP-1 expres- sion is not detectable [2], [17] or very low [18] in healthy young adult mice, but is found in high concentrations in the eyes of CNV bearing mice with the infiltration of macrophages [2], [17], [19], [20]. Recent studies also demonstrate an association between elevated urinary MCP-1 level and AMD [21], and the intraocular PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e28933