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Priyatham S Mettu, Peter Saloupis, Scott W Cousins; PAMP Stimulation of Macrophages Promotes Neovascular Remodeling in Experimental Choroidal Neovascularization. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1198.
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© ARVO (1962-2015); The Authors (2016-present)
Neovascular remodeling (NVR), the transformation of capillaries into branching arterioles with perivascular fibrosis, is a major cause of persistent disease activity in spite of anti-VEGF therapy in NV AMD. Pathogen-associated molecular patterns (PAMPs) are microbe-associated molecules that can stimulate activation of macrophages via binding to cell-surface pattern-recognition receptors. We explored the hypothesis that PAMP stimulation of macrophages promotes NVR in experimental laser-induced choroidal neovascularization (CNV).
We performed laser-induced CNV in wild-type C57BL/6J mice (8 mo. old, n=18). At the time of laser, half of these mice (n=9) underwent PAMP stimulation with low-dose lipopolysaccharide (10 μg, well below dose associated with systemic toxicity), while the other half (n=9) received saline control. After two weeks, fluorescein angiography (FA) was performed, and eyes were removed and processed for choroidal flatmount and immunohistochemistry. Macrophage activation following PAMP stimulation was assessed by RT-PCR of select cytokines in isolated splenic monocytes.
PAMP-stimulated mice demonstrated a mean lesion size of 3.7 ± 0.8 disc areas (DA) by propidium iodide cellular flatmount, as compared to 2.0 ± 0.7 DA for control exposed mice (p < 0.01). PAMP-stimulated mice also demonstrated larger lesions by TRITC-lectin vascular morphology flatmounts, 2.3 ± 0.3 DA, as compared to 1.4 ± 0.1 DA for control mice (p < 0.02). NVR was readily observed in CNV of PAMP-stimulated mice and was characterized by prominent leakage by FA as well as large-caliber branching arterioles with vascular loops on vascular morphology flatmounts. In contrast, saline-exposed mice had small-caliber capillaries with few arterioles, as well as mild FA leakage. Preliminary studies suggest that PAMP-stimulated mice demonstrate increased ratio of SMA+ smooth muscle cells / CD31+ endothelial cells, as well as increased F4/80+ macrophage frequency. Additionally, splenic monocytes from PAMP-stimulated mice had increased activation state, with upregulated expression of cytokines potentially relevant to NVR, including NOS-2, MMP-9, and PDGF.
PAMP-stimulation of macrophages appears to promote NVR in experimental CNV. Importantly, this highlights a potential role for therapies directed against activated macrophages in the treatment of persistent disease activity in NV AMD.
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