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Shira Levi, Michelle Grunin, Sarah Elbaz-Hayoun, Batya Rinsky, Itay Chowers; Pro-Angiogenic Mechanism of Activated Macrophages from Patients with Age-related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1628.
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Monocytes/macrophages may exert a pro-angiogenic effect in the context of neovascular age-related macular degeneration (nvAMD). We have previously found a pro-angiogenic effect of polarized M(IFNγ and LPS) human macrophages from nvAMD patients as compared to age-matched controls. This effect was identified in-vivo in the rat model of laser-induced choroidal neovascularization (LI-CNV), with a correlation to the ex-vivo choroid sprouting assay (CSA) findings. We now aim to explore the mechanism which mediates the pro-angiogenic effect of the activated macrophages from nvAMD patients.
Monocytes were isolated from nvAMD patients and were differentiated into Mo, M(IFNγ and LPS), and M(IL-13+IL-4) macrophages. Protein levels of candidate cytokines for mediation of angiogenic effects were assessed in the macrophages' cell culture supernatants using ELISA. To assess the macrophages' pro-angiogenic mechanism, we performed CSA, initially with macrophages' supernatants, and then with the addition of candidate cytokines which were differentially secreted by the activated macrophages.
A higher CSA sprouting area (SA) was identified following addition of media from M(IFNγ and LPS) cells as compared to non-treated wells (mean of ratios±SEM 3.62±0.52, P =0.0001; t-test; n=19). Mo and M(IL-13+IL-4) cells' supernatant had no effect on SA (1.57±0.34, p=0.11; n=19, and 1.55±0.5, p=0.28; n=14, respectively). TNFα (5.4-fold, p=0.0001), VEGFa (1.5-fold, p=0.007), and IL-6 (11.2-fold, p=0.0001) were up-regulated in the pro-angiogenic M(IFNγ and LPS) macrophage phenotype as compared to Mo. Cytokines at their levels in the M(IFNγ and LPS) culture media per ELISA, were added to the CSA culture. VEGFa (0.86±0.08, p=0.16; n=9), and IL1β (0.8±0.2, p=0.34; n=10) showed no effect on the SA as compared to control wells, while TNFα was associated with enlarged SA (1.6±0.2, p=0.01; n=8,), and IL-6 (0.64±0.1, p=0.01; n=9) and IL-8 (0.47±0.14, p=0.007; n=8) with decreased SA.
These data further supports the putative role of macrophages and their cytokine products in modulating CNV. It also suggests that factors other than VEGF may also mediate such a pro-angiogenic macrophage effect, while other cytokines may suppress CNV. Thus, targeting multiple cytokines simultaneously may potentially serve as a therapeutic strategy for the disease.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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