CCR2 Blocker Inhibits Choroidal Neovascularization Figure 4. Phosphorylated extracellular signal-regulated kinase (p-ERK1/2) expression in Western blot. (A) A representative blot. p-ERK expression in the choroid-RPE complex from vehicle-treated mice and INCB3344-treated mice on Day 3 after laser photocoagulation, and normal mice (without photocoagulation). Western blot analysis revealed that p-ERK expression increased after laser photocoagulation and was suppressed by INCB3344 treatment. (B) Semi-quantitative analysis of the band intensity showed an increase in relative p-ERK expression (values normalized to total ERK expression) in the eyes of photocoagulated mice compared with untreated mice(n = 8, *P,0.001; n = 8, **P,0.001), and INCB3344 suppressed this increase (n = 8, ***P,0.001). doi:10.1371/journal.pone.0028933.g004 vascular tissue comprising the vessel lumen, but the INCB3344- treated mice had smaller lesion areas comprising fibro-vascular tissue, disturbed RPE and pigment clumps compared with the vehicle-treated mice (Fig. 1A). Choroid-RPE flat mounts confirmed the distinct reduction in CNV area by the INCB3344 treatment (Fig. 1 B). At Day 14 after photocoagulation, the mean CNV area was 19,759.56861.1 mm2 in vehicle- treated mice (n = 78 spots), which significantly decreased in INCB3344-treated mice (11,392.26468.8 mm2, n = 81 spots). This translated into a 42.4% decrease in CNV area by INCB3344 treatment (P,0.001) (Fig. 1C). Inhibition of macrophage infiltration by INCB3344 treatment The number of F4/80 positive cells was substantially lower in INCB3344-treated mice than in vehicle-treated mice in both choroid- RPE flat mounts (Fig. 2A) and sections (Fig. 3A). In the flow cytometry quantitative analyses, we detected only a few macrophages in normal mice (without laser treatment). On Day 3 after laser treatment, the number of macrophages dramatically increased in both vehicle- and INCB3344-treated mice, but INCB3344 treatment significantly suppressed the number of macrophages compared to vehicle treatment (Fig. 2B. n = 5, P,0.001). Decrease of VEGF expression by INCB3344 treatment Strong VEGF-positive immunoreactivity was detected in the laser injury sites. The immunoreactivity was mainly localized to infiltrating macrophages (F4/80 positive cells) at the laser injury site. INCB3344 treatment decreased the VEGF immunostaining compared to vehicle treatment (Fig. 3A). ELISA showed that VEGF protein levels in the choroid-RPE complex in INCB3344-treated mice were significantly lower (199.168.2 pg/mg) compared with vehicle-treated mice (289.3627.8 pg/mg, P = 0.012, n = 8) (Fig. 3B). Real-time PCR analysis on sorted macrophages from choroid- RPE complexes showed that VEGF mRNA expression signifi- cantly increased in those infiltrating macrophages after photoco- agulation (P,0.001, n = 3), but it was markedly suppressed by INCB3344 treatment (P,0.001, n = 3) (Fig. 3C). Suppression of phosphorylated ERK1/2 (p-ERK1/2) by INCB3344 treatment Phosphorylation of ERK1/2 is considered a measure of MAPK activation, which regulates a variety of angiogenic factors including VEGF. The inhibitory effects of INCB3344 on ERK1/2 phosphorylation in response to MCP-1 stimulation via CCR2 have been shown in vitro [26] [27]. Here, we further investigate the effect of INCB3344 on ERK1/2 phosphorylation in a CNV animal model. On Day 3 after photocoagulation, the p-ERK1/2 expression level increased relative to that of total ERK1/2 in the choroid- RPE complex in both vehicle- and INCB3344-treated mice compared with baseline levels (untreated normal mice), while the relative expression of p-ERK1/2 was significantly reduced in the INCB3344-treated mice compared with the vehicle-treated mice (n = 8, P,0.001) (Fig. 4). Regression of established CNV by INCB3344 treatment As previously reported, senescent Sod12/2 mice have many features in common with patients with AMD, such as sub-RPE deposits, thickened Bruch’s membrane and CNV [32]. Spontaneous CNVs were detected in 13 Sod12/2 mice by both fundus examination and fluorescein angiography. INCB3344 treatment caused a regression in established CNV (Fig. 5A). The size of CNV significantly decreased from 2971.861976.3 mm2 (range from 172.469.2 mm2 to 12661.0669.2 mm2, n = 6) before treatment to 1267.96861.2 mm2 (range from 0 to 5452.3674.4 mm2) at 1 week after treatment (P,0.001). Among them, two CNVs with the minimum sizes (172.469.2 mm2 and 219.769.8 mm2) were com- pletely abolished by the INCB3344 treatment, which was confirmed by histological examinations in serial cryo-sections (Data not shown). On the other hand, in vehicle-treated mice, no significant CNV regression was detected. CNV size was 1876.76709.3 mm2 (range from 260.68615.22 mm2 to 5466.466.44 mm2, n = 7) before treatment and 1872.26707.6 mm2 (range from 249.606 7.19 mm2 to 5450.16632.17 mm2) after treatment. Paired pre-and post-comparison of CNV size showed that INCB3344 treatment caused a 70.3569.86% decrease (n = 6) in CNV size (Fig. 5B), which was significantly higher than that in vehicle treatment (161%, n = 7, P,0.001). PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e28933