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Ali Hafezi-Moghadam, Souska Zandi, Shintaro Nakao, Dawei Sun, Sonja Frimmel, Zhongyu Zhang, Tatsuro Ishibashi; Selective ROCK2 inhibition causes an M1-macrophage shift in choroidal neovascularization. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):843.
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© ARVO (1962-2015); The Authors (2016-present)
Plasticity and diversity are fundamental characteristics of macrophages. Undifferentiated M0 macrophages polarize into the classical pro-inflammatory M1-like and the alternative anti-inflammatory M2-like macrophages. Macrophage plasticity in age-related macular degeneration (AMD) is not well understood. Here, we introduce a novel role for ROCK2 in macrophage plasticity and its impact on ocular immune balance.
CNV was induced in C57BL/6J mice using a 532-nm laser (100mW, 50µm, 100ms). Western Blot was performed for CD206, CCR7, CD80 and β-tubulin . Choroids were harvested at different time points (4 h, 1, 3, 7 and 14 days) after CNV induction. On day 7 mice were treated with dual ROCK1/2- and a ROCK 2 selective inhibitor. For flow cytometry, retinal and choroidal macrophages were prepared from mouse eyes for flow cytometry. After laser injury, eyes were enucleated at different time points (1, 2, 3, 5, and 7days) and stained for CD11b-PE, CD80-FITC, CD206-FITC, or isotype control.
The number of CD11b(+)CD80(+) M1-like macrophages increased on day 1 after laser injury and remained high through day 7 (n=6 animals, P<0.01). We found a peak of CD11b(-)CD206(+) cells on day 2 post laser injury, which preceded the reported start of angiogenesis. On days 3 through 7 the percentage of CD11b(+)CD206(+) cells increased with a peak on day 7, coinciding with the maximum angiogenic response in the laser-injury model (n=6 animals, P<0.01). Dual ROCK and selective ROCK2 inhibition substantially decreased the CD11b(+)CD206(+) M2 population, when examined on day 7, while ROCK2 inhibition increased the percentage of CD80(+) cells. Western blot showed that ROCK2 inhibition but not dual ROCK1/2 inhibition increased CD80 and CCR7 in lasered eyes.
Selective ROCK2 inhibition increases M1-like macrophage markers, CD80 and CCR7. Targeting macrophage plasticity through ROCK signaling preserves the beneficial macrophages that are essential for retinal health and simultaneously restores the balance between pro-angiogenic macrophages and their angiostatic counterparts.
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