Suppression and regression of choroidal neovascularization in mice by a novel CCR2 antagonist, INCB3344

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 µm-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 Sod1⁻/⁻ 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.

Figure 1. Effect of INCB3344 on CNV formation.

(A) Haematoxylin–eosin-stained light micrograph of CNV lesions on Day 14 after laser photocoagulation. Each photograph shows the central area of CNV lesions in vehicle-treated or INCB3344-treated mice. Scale bar = 100 µm. (ILM: internal limiting membrane; NFL: nerve fiber layer; GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; IS: inner segment; OS: outer segment; RPE: retinal pigment epithelium; C: choroid; S: sclera). (B) Representative micrographs of CNV lesions in the choroid-RPE flat mounts from laser-induced CNV in mice treated with vehicle or INCB3344. CNV areas were perfused with fluorescein isothiocyanate-dextran in flat-mount choroid-RPE complex. Scale bar = 100 µm. (C) Quantitative analysis of CNV size. Values are mean ± SE, vehicle, n = 78 spots, INCB3344, n = 81 spots. *P<0.001.

Figure 2. Macrophages detected by immunohistochemistry of choroid-RPE flat mounts and flow cytometry.

(A) Immunohistochemistry of macrophages in choroid-RPE flat mounts on Day 3. After photocoagulation, a large number of macrophages accumulated at the laser injury sites. INCB3344 suppressed this increase. Scale bar = 100 µm. (B) Left: Overlay histogram of flow cytometric results. Right: Flow cytometric analysis data with F4/80 staining of the macrophages in choroid-RPE on Day 3 after laser photocoagulation (Macrophage numbers per choroid-RPE complex). After photocoagulation, the number of macrophages significantly increased compared with no laser photocoagulation controls (relative to normal control, ^* P<0.001 n = 5, ^** P<0.001 n = 5). INCB3344 treatment significantly reduced the number of macrophages compared to the vehicle-treated group (^*** P<0.001, n = 5).

Figure 3. VEGF expression.

(A) Immunohistochemistry of macrophages (green) and VEGF (red) in cryosections on Day 3. Significantly higher levels of VEGF were expressed in macrophages at the photocoagulated sites. VEGF localized mainly in infiltrating macrophages at the laser injury sites. INCB3344 apparently decreased VEGF immunoreactivity compared to vehicle treatment. The negative control omitting the primary antibody (second antibody only) had detectable auto-fluorescence in RPE. Scale bar = 100 µm. (B) VEGF protein levels in the choroid-RPE were quantitatively measured by ELISA. VEGF levels on Day 3 were significantly suppressed by INCB3344 treatment. (n = 8, *P = 0.012). (C) The expression of VEGF mRNA derived from macrophages isolated from choroid-RPE complexes was detected by real-time PCR on Day 3 after photocoagulation. The increased VEGF mRNA expression in infiltrating macrophages was significantly suppressed by INCB3344 treatment (**,***P<0.001, n = 3).

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).

Figure 5. Effect of INCB3344 on established CNV.

(A) Fundus photographs (1^st and 3^rd rows) and fluorescent angiography (2^nd and 4^th rows) pre-treatment (1^st column) and post-treatment (2^nd column) with vehicle (top 2 rows) or INCB3344 (bottom 2 rows). Established CNV in Sod1 ^−/− mice were markedly regressed by INCB3344 treatment (bottom), while no significant regression of established CNV was detected in vehicle treatment (top). Scale bar = 20 µm. (B) INCB3344 treatment caused a 70.35±9.86% decrease (n = 6) in established CNV size, which was significantly higher than that in vehicle treatment (1.0±1.0%, n = 7, *P<0.001).