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Priyatham S Mettu, Peter Saloupis, Daniel Saban, Scott W Cousins; Increased Frequency of Nonclassical Monocytes in the Blood is Correlated with Development of Neovascular Remodeling in Experimental Choroidal Neovascularization. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2311.
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© ARVO (1962-2015); The Authors (2016-present)
In experimental laser choroidal neovascularization (CNV), neovascular remodeling (NVR) is characterized by transformation of small lesions with capillaries into large lesions with arterioles and perivascular fibrosis. We have shown that blood-derived macrophages mediate NVR. We hypothesize that the key blood monocyte subset that mediates NVR is the nonclassical Ly6Clo “reparative” subset rather than the classical Ly6Chi “inflammatory” subset. We sought to provide supportive evidence for this hypothesis in a model of pathogen-associated molecular pattern (PAMP) stimulation NVR.
Laser CNV was induced in wild-type C57BL6/J mice (7-9 mo. old) exposed to low-dose of the PAMP, lipopolysaccharide (LPS) (10 μg, well below systemic toxic dose) or PBS control. Half of the mice in each group were euthanized at 3 days post-laser, the time point when capillary to NVR transition begins. Blood and eyes were recovered to assess effects of PAMP exposure on blood monocyte subset frequency and CNV macrophage infiltration, by flow cytometry and immunohistochemistry, respectively. In the other half of mice, eyes were recovered for choroidal flatmount analysis and immunohistochemistry to assess for NVR.
LPS-exposed mice developed NVR, defined by CNV with increased SMA+ arterioles and collagen IV deposition, and demonstrated increased frequency of F4/80+ macrophage CNV infiltration, as compared to control mice. Presence of NVR was associated with a major change in the frequency of nonclassical to classical monocytes in the blood. LPS-exposed mice with NVR: 36% nonclassical Ly6Clo to 64% classical Ly6Chi blood monocytes; PBS-exposed mice with capillary lesions: 11% nonclassical Ly6Clo to 89% classical Ly6Chi blood monocytes (p <0.01). Further, LPS exposure produced a major change in absolute number of Ly6Clo blood monocytes: LPS-exposed mice - 4978 cells / μL of blood versus PBS-exposed mice - 727 cells / μL (p < 0.01).
Development of NVR in experimental CNV appears to occur in association with increased frequency of circulating nonclassical blood monocytes, suggesting that NVR may be mediated by increased infiltration of nonclassical monocytes at the site of incipient CNV formation. Additional experiments will assess alterations in CNV macrophage subsets and in expression of fibrogenic cytokines, in the setting of NVR.
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