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Souska Zandi, Shintaro Nakao, Ryoichi Arita, Yasuaki Hata, Alexander Schering, Dawei Sun, Sonja Frimmel, Mitsuko Yuzawa, Tatsuro Ishibashi, Ali Hafezi-Moghadam; Rho Kinase Mediated Macrophage Polarization in AMD. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2292.
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Choroidal neovascularization (CNV) is the main cause of severe vision loss in patients with age-related macular degeneration (AMD). Macrophages are involved in the pathology of AMD. However, the function of ocular infiltrating macrophages and their subtypes in AMD is not fully understood. We introduce the role of Rho/Rho kinase (ROCK) signaling pathway in macrophage recruitment and polarization in AMD.
Human sections were stained with antibodies (Abs) against human CD206 (MMR), or human ROCK1 or ROCK2. To examine macrophages in the retina and choroids in vivo, cells were prepared from mouse eyes. To collect a sufficient number of ocular infiltrating cells, 50 burns were delivered to mouse eyes by laser. The eyes were enucleated at 1, 2, 3, 5, and 7days after laser injury. The anterior segment (cornea, iris, and lens) was excised and the posterior segment of the eye including sclera, choroid, and retina was disrupted. The supernatants were collected and passed through a mesh and viable cells were obtained. A total of 12 eyes (6 individual pools) were examined per group. The cells were stained with PE anti-mouse CD11b, FITC anti-mouse CD206 (MMR) and PE-Cy5 anti-mouse CD80 Abs. Our novel ROCK2 inhibitor or vehicle was injected intraperitoneally every day.
In AMD membranes from human patients, ROCK1 and 2 colocalized with CD206, which is strongly upregulated on pro-angiogenic M2-like macrophages. We isolated cells from mouse CNV membranes and found that there were significantly more infiltrated CD11b(+) cells in the lasered eyes on days 1 and 5; CD11b(+)CD206(-) macrophages in CNV eyes were more than control eyes at all time points. he number of CD11b(+)CD80(+) M1-like macrophages significantly increased on day 1 after laser injury and remained high through day 7. In contrast, the number of CD11b(+)CD206(+) M2-like macrophages was only significantly increased on day 7 after laser injury. OCK2 inhibition substantially decreased the CD11b(+)CD206(+) M2 population, when examined on day 7.
This work elucidates a to-date unknown role of rho kinase in macrophage polarization and establishes the importance of new M2-like macrophage phenotype in AMD pathology. Rho kinase may thus become an attractive molecular target in the prevention and treatment of AMD.
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