Abstract
Objective
Macrophages show extreme heterogeneity and different subsets have been characterized by their activation route and their function. For instance, macrophage subsets are distinct by acting differently under pathophysiological conditions such as inflammation and cancer. Macrophages also contribute to angiogenesis, but the role of various specific subsets in angiogenesis has not been thoroughly investigated.
Methods and results
Matrigel supplemented with macrophage subsets [induced by IFNγ (M1), IL-4 (M2a) or IL-10 (M2c)] was injected subcutaneously in C57BL/6 J mice and analyzed by CD31 staining after 14 days. Increased numbers of endothelial cells and tubular structures were observed in M2-enriched plugs compared to control and other subsets. Additionally, more tubular structures formed in vitro in the presence of M2 macrophages or their conditioned medium. To identify a mechanism for the pro-angiogenic effect, gene expression of angiogenic growth factors was analyzed. Induced expression of basic fibroblast growth factor (Fgf2), insulin-like growth factor-1 (Igf1), chemokine (C–C motif) ligand 2 (Ccl2) and placental growth factor (Pgf) was observed in M2 macrophages. Using a blocking antibody of PlGF to inhibit M2c induced angiogenesis resulted in mildly reduced (40 %) tube formation whereas neutralization of FGF-2 (M2a) signaling by sFGFR1-IIIc affected tube formation by nearly 75 %.
Conclusions
These results indicate that macrophages polarized towards an M2 phenotype have a higher angiogenic potential compared to other subsets. Furthermore, we propose FGF signaling for M2a- and PlGF signaling for M2c-induced angiogenesis as possible working mechanisms, yet, further research should elucidate the exact mechanism for M2-induced angiogenesis.
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Acknowledgments
This work was supported by the Netherlands Heart Foundation (Dr. E Dekker post-doctoral fellow Grant [Grant Numbers 2007T034, 2012T079] to Dr. Donners; Dr. E Dekker Established Investigator Grant [Grant Number 2007T067] and NWO-VIDI Grant [Grant Number 917.066.329] to Dr. de Winther). Dr Post is supported by Grants from BMM (PENT, iValve) and CTMM/Netherland Heart Foundation (EMINENCE): These research programs of the BioMedical Materials institute and the Center for Translational and Molecular Medicine are co-funded by the Dutch Ministry of economic affairs Agriculture and Innovation.
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10456_2013_9381_MOESM1_ESM.tif
Supplemental figure 1. Gene expression profile of macrophage subsets after 24h polarization. After 8 days of culturing, macrophages are polarized with IFNγ, IL-4 or IL-10 (n=3). A, IFNγ stimulation results in increased expression of Nos2, Tnfa and Il10 (C). B, IL-4 stimulation results in upregulation of Chi3l3, Arg1 and Mrc1 (C) whereas IL-10 stimulates expression of Il10 and Mrc1(C). The statistical significance was determined by one-way ANOVA. **p<0.01. (TIFF 71 kb)
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Supplemental figure 2. Fluorescently labeled macrophages (red) and endothelial cells (green) in a Matrigel plug. A, Co-localization of macrophages and endothelial cells is apparent in vivo. B, Microvessels are surrounded by macrophages. (TIFF 151 kb)
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Supplemental figure 3. Inhibition of PlGF signaling using soluble Flt-1 in a tube formation assay. sFlt-1 reduced tube formation of endothelial cells alone (-M, 34.7%), endothelial cells co-cultured with M0 macrophages (27.4%) or M2c macrophages (44.5 %) compared to control. The data represent the mean ± SEM. The statistical significance was determined by one-way ANOVA. (TIFF 39 kb)
10456_2013_9381_MOESM4_ESM.tif
Supplemental figure 4. ELISA for PlGF. Concentration of PlGF protein was measured in concentrated conditioned medium of macrophage subsets. A trend could be observed towards more PlGF production in M2c macrophages. The data represent the mean ± SEM. (TIFF 35 kb)
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Jetten, N., Verbruggen, S., Gijbels, M.J. et al. Anti-inflammatory M2, but not pro-inflammatory M1 macrophages promote angiogenesis in vivo. Angiogenesis 17, 109–118 (2014). https://doi.org/10.1007/s10456-013-9381-6
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DOI: https://doi.org/10.1007/s10456-013-9381-6