Figure 5. Effect of INCB3344 on established CNV. (A) Fundus photographs (1st and 3rd rows) and fluorescent angiography (2nd and 4th rows) pre-treatment (1st column) and post-treatment (2nd column) with vehicle (top 2 rows) or INCB3344 (bottom 2 rows). Established CNV in Sod12/2 mice were markedly regressed by INCB3344 treatment (bottom), while no significant regression of established CNV was detected in vehicle treatment (top). Scale bar = 20 mm. (B) INCB3344 treatment caused a 70.3569.86% decrease (n = 6) in established CNV size, which was significantly higher than that in vehicle treatment (1.061.0%, n = 7, *P,0.001). doi:10.1371/journal.pone.0028933.g005 CCR2 Blocker Inhibits Choroidal Neovascularization Discussion In the present study, we evaluated the therapeutic value of INCB3344, a CCR2 antagonist, on a mouse model of age-related macular degeneration and demonstrated that INCB3344 treat- ment markedly suppressed new CNV formation and shrank established CNVs. To investigate the possible cellular mechanism of INCB3344 suppression of CNV, we evaluated the infiltration of macrophag- es in the early phase of laser induced CNV and revealed that macrophage infiltration was significantly suppressed by INCB 3344 treatment. In this study, we evaluated the macrophage infiltration on Day 3, the day of peak macrophage response [2], [13], [33]. We delivered INCB3344 to the vitreous cavity immediately after photocoagulation. After photocoagulation, local MCP-1 increases quickly [33], and recruits monocytes [34], [35] to laser injury sites, where they become inflammatory macrophages [36], [37]. Acting as a CCR2 antagonist, INCB3344 has displayed a high ability to inhibit monocyte chemotaxis in vitro and suppress macrophage influx in a variety of preclinical animal models of inflammatory diseases [26], [27]. Moreover, recent research reports that topical treatment of a CCR2 antagonist leads to the blocking of CCR2/MCP-1 interaction and reduces monocyte infiltration into the cornea in the dry eye disease mouse model [38]. These data are consistent with our results, which demonstrate that the suppression of monocyte/macrophage infiltration acts as an important cellular mechanism for INCB3344 treatment in the current model. To determine the impact of INCB3344 on the downstream signaling of macrophages in CNV formation, we detected VEGF on the peak response day of macrophages. Our results demon- strated that VEGF significantly decreased with the suppression of infiltrating macrophages by INCB3344 treatment. After photoco- agulation, VEGF is up-regulated [10], [11], [33] and acts as a promoting mediator in the development of CNV [11], [39], [40]. The variation of VEGF levels correlates highly to that of macrophages after laser injury [25], and their peak responses are reported to coincide with each other [13], [33]. While in pharmacologically macrophage-depleted mice [12], VEGF pro- duction is reduced in proportion to the decrease in the number of macrophages. Moreover, enriched ocular-infiltrating macrophages from laser-induced model mice have shown angiogenic ability in a dorsal air sac assay, and express activation-surface markers (class II, CD40, B7-1 and B7-2 molecules) and the mRNA for potential angiogenic factors including VEGF [16], which indicates that the infiltrating macrophages are a rich source of VEGF. Our results agree with these data and reveal that macrophages play an important role in the variation of intro-ocular VEGF after laser injury. Further more, our results demonstrate that elevated VEGF expression in infiltrating macrophages is suppressed by INCB3344. We reveal that INCB3344 can not only inhibit macrophage infiltration but also suppress the angiogenic ability of infiltrating macrophages, which results in the reduction of VEGF, and finally in suppression of CNV. In this study, we revealed that INCB3344 treatment inhibited CNV formation via the suppression of macrophage infiltration. Our study focused on CCR2, macrophages, and VEGF, although several other cytokines such as tumor necrosis factor-alpha (TNF- a) [41]–[][43], interleukin -1beta (IL-1b) [41], [43], hypoxia inducible factors (HIF-1a and HIF-2a) [44], IL-6 [35] and tissue factor (TF) [5] are reported to be involved in CNV formation. We cannot rule out a potential link between macrophages and other cytokines, however, VEGF is crucial in the pathogenesis of CNV formation because anti-VEGF drug therapy, for example, PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e28933