bevacizumab and ranibizumab have achieved an obvious effect in CNV due to age-related macular degeneration. We demonstrated that the ERK1/2 phosphorylation induced by laser treatment was significantly suppressed by INCB3344. We examined the phosphorylation of ERK 1/2 because the activation of ERK1/2 in macrophages can be induced through the CCR2 [45] and their activation is thought to be a key component in the cellular events leading to the infiltration and activation of macrophages [46]–[][48]. Our results are consistent with the results in vitro [20], [21] and the previous reports that blockage of CCR2 by anti-CCR2 monoclonal antibodies inhibits phosphorylation of ERK1/2 in peritoneal macrophages [45]. Our results on the inhibitory effect on VEGF secretion are also compatible with the data that ERK1/2 activation in macrophages [49] or monocytes [34] is reported to be responsible for VEGF production in these cells. We can conclude that INCB3344 inhibits the activation of ERK1/2 in macrophages by blocking CCR2, which results in the reduction of macrophage infiltration and VEGF production. We further evaluated the therapeutic effect of INCB3344 on established CNV in Sod12/2 mice. SOD1 is a component of the antioxidant defense system of the retina, and it has been demonstrated that SOD1 deficiency leads to retinal dysfunction and progressive, degenerative changes of retinal cell layers [50]. Senescent Sod12/2 mice have many features in common with patients with AMD, such as age-related accumulation of sub-RPE deposits, thickened Bruch’s membrane, and spontaneous CNV, which recapitulates the key elements of the human pathology [32]. In this study, we detected the established spontaneous CNV by screening senescent Sod12/2 mice, and then investigated the therapeutic effect of INCB3344 on this CNV model. This process can closely mimic the diagnosis and treatment of an AMD patient with CNV. Although the size of these CNV varied in the Sod12/2 mice, by paired pre- and post-comparison, we demonstrated that INCB3344 treatment caused marked regression of established CNV. In our results, fluorescence leakage in CNV area significantly reduced or disappeared in FA by the treatment of INCB3344, whereas the lesion area did not show much References 1. Fine SL, Berger JW, Maguire MG, Ho AC (2000) Age-related macular degeneration. N Engl J Med 342: 483–492. 2. Yamada K, Sakurai E, Itaya M, Yamasaki S, Ogura Y (2007) Inhibition of laser- induced choroidal neovascularization by atorvastatin by downregulation of monocyte chemotactic protein-1 synthesis in mice. Invest Ophthalmol Vis Sci 48: 1839–1843. 3. Grossniklaus HE, Green WR (2004) Choroidal neovascularization. Am J Ophthalmol 137: 496–503. 4. Tsutsumi-Miyahara C, Sonoda KH, Egashira K, Ishibashi M, Qiao H, et al. (2004) The relative contributions of each subset of ocular infiltrated cells in experimental choroidal neovascularisation. Br J Ophthalmol 88: 1217–1222. 5. Grossniklaus HE, Ling JX, Wallace TM, Dithmar S, Lawson DH, et al. (2002) Macrophage and retinal pigment epithelium expression of angiogenic cytokines in choroidal neovascularization. Mol Vis 8: 119–126. 6. Kamei M, Yoneda K, Kume N, Suzuki M, Itabe H, et al. (2007) Scavenger receptors for oxidized lipoprotein in age-related macular degeneration. Invest Ophthalmol Vis Sci 48: 1801–1807. 7. Ferrara N, Davis-Smyth T (1997) The biology of vascular endothelial growth factor. Endocr Rev 18: 4–25. 8. Xiong M, Elson G, Legarda D, Leibovich SJ (1998) Production of vascular endothelial growth factor by murine macrophages: regulation by hypoxia, lactate, and the inducible nitric oxide synthase pathway. Am J Pathol 153: 587–598. 9. Yi X, Ogata N, Komada M, Yamamoto C, Takahashi K, et al. (1997) Vascular endothelial growth factor expression in choroidal neovascularization in rats. Graefes Arch Clin Exp Ophthalmol 235: 313–319. 10. Ishibashi T, Hata Y, Yoshikawa H, Nakagawa K, Sueishi K, et al. (1997) Expression of vascular endothelial growth factor in experimental choroidal neovascularization. Graefes Arch Clin Exp Ophthalmol 235: 159–167. 11. Espinosa-Heidmann DG, Suner IJ, Hernandez EP, Monroy D, Csaky KG, et al. (2003) Macrophage depletion diminishes lesion size and severity in experimental choroidal neovascularization. Invest Ophthalmol Vis Sci 44: 3586–3592. CCR2 Blocker Inhibits Choroidal Neovascularization amelioration in fundus photographs. This discrepancy may be due to the accumulation of irreversible histological damage in the CNV area including sub-RPE deposits or fibrosis, and future investigations with more samples for longer duration, and evaluation with optical coherence tomography may be helpful. In this study, we showed that blockage of the CCR2/MCP- 1(CCL2) signal pathway by INCB3344 suppressed the develop- ment of CNV in laser induced model. However, there is some controversy over the issue of CNV formation associated with the CCR2/MCP-1 signal pathway. Ambati et al. [51] detected spontaneous CNV in senescent Ccl-2 (MCP-1) KO mice and Ccr-2 KO mice, while Luhmann et al. [30] were unable to detect any spontaneous CNV in similarly aged Ccl-2 KO mice and revealed that the AMD-like features in MCP-1 KO mice described by Ambati et al. might be the result of aging alone. Although these discrepancies need further investigation, the size of laser induced CNV was reduced in MCP-1 KO mice [30] and Ccr-2 KO mice [16] with fewer macrophages compared with wild type mice. The results of these reports are consistent with our conclusion that suppression of macrophage migration via blockage of CCL-2/ CCR-2 with INCB3344 inhibits CNV formation in a laser- induced CNV model. 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(2008) Vascular adhesion protein-1 blockade suppresses choroidal neovascularization. FASEB J 22: 2928–2935. 20. Satofuka S, Ichihara A, Nagai N, Noda K, Ozawa Y, et al. (2008) (Pro)renin receptor promotes choroidal neovascularization by activating its signal transduction and tissue renin-angiotensin system. Am J Pathol 173: 1911–1918. 21. Guymer RH, Tao LW, Goh JK, Liew D, Ischenko O, et al. (2011) Identification of urinary biomarkers for age-related macular degeneration. Invest Ophthalmol Vis Sci 52: 4639–4644. 22. Jonas JB, Tao Y, Neumaier M, Findeisen P (2010) Monocyte chemoattractant protein 1, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 in exudative age-related macular degeneration. Arch Ophthalmol 128: 1281–1286. PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e28933