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D. G. Espinosa-Heidmann, G. Malek, K. Wu, P. Saloupis, S. W. Cousins; Osteopontin Regulation of Choroidal Neovascular Fibrosis Through Macrophages. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6171.
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Macrophages are observed in all stages of age-related macular degeneration. Our studies have shown that (1) these macrophages are mostly blood-derived monocytes recruited to the choriocapillaris and (2) the pre-existing activation state of the recruited macrophages determines the impact, good or bad, on experimental choroidal neovascularization (CNV) severity. Signals involved in recruitment and retention of macrophages within the tissue are unknown. Osteopontin (OPN) is a possible macrophage retention factor recently identified in several degenerative diseases of aging. OPN secreted by smooth muscle cells may serve to localize macrophages adjacent to maturing vessels. We hypothesized that vascular remodeling and maturation observed in patients with severe CNV is driven in part by OPN secreted by mesenchymal cells, which act to retain macrophages adjacent to these cells.
Laser CNV was induced in young (2 month old, n=6) and old (14-16 month old, n=5) OPN knockout and wild type C57BL/6 mice. Three weeks post laser induction mice were sacrificed, the right eye was collected and processed for flatmounts to evaluate the vascularity, cellularity and size of the CNV lesion and the left eye was cryoprotected and processed for immunohistochemistry. Cryosections were probed with markers for macrophages (F48-80 and Iba-1), mesenchymal (smooth muscle-actin) and enodothelial (von willebrand factor) cells as well as OPN. Number of immunopositive cells within the lesion were counted and compared in young versus old mice.
CNV lesions in old mice were significantly larger and more fibrotic than those found in young mice regardless of genotype. OPN expression was observed surrounding developing blood vessels and within fibrotic zones of CNV of both young and old wildtype mice. In old OPN knockout mice, CNV size was reduced compared to age-matched wildtype mice. Also, CNV from knock out mice demonstrated an approximately 40% decrease in the number of F4-80 positive macrophages and 36% decrease in vascular smooth muscle cells compared to wildtype controls.
Aged OPN knockout mice develop smaller and less fibrotic CNV compared to wildtype mice, which correlates with decreased macrophage infiltration and vascular smooth muscle cells. These data suggest that OPN may play a role in recruitment and retention of macrophages in the laser CNV.
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