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Anti-inflammatory M2, but not pro-inflammatory M1 macrophages promote angiogenesis in vivo

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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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References

  1. Virmani R et al (2005) Atherosclerotic plaque progression and vulnerability to rupture: angiogenesis as a source of intraplaque hemorrhage. Arterioscler Thromb Vasc Biol 25(10):2054–2061

    Article  CAS  PubMed  Google Scholar 

  2. Khurana R et al (2005) Role of angiogenesis in cardiovascular disease: a critical appraisal. Circulation 112(12):1813–1824

    Article  PubMed  Google Scholar 

  3. Moore KJ, Tabas I (2011) Macrophages in the pathogenesis of atherosclerosis. Cell 145(3):341–355

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Sluimer JC et al (2008) Hypoxia, hypoxia-inducible transcription factor, and macrophages in human atherosclerotic plaques are correlated with intraplaque angiogenesis. J Am Coll Cardiol 51(13):1258–1265

    Article  CAS  PubMed  Google Scholar 

  5. Xue L, Greisler HP (2002) Angiogenic effect of fibroblast growth factor-1 and vascular endothelial growth factor and their synergism in a novel in vitro quantitative fibrin-based 3-dimensional angiogenesis system. Surgery 132(2):259–267

    Article  PubMed  Google Scholar 

  6. Xiong M et al (1998) Production of vascular endothelial growth factor by murine macrophages: regulation by hypoxia, lactate, and the inducible nitric oxide synthase pathway. Am J Pathol 153(2):587–598

    Article  CAS  PubMed  Google Scholar 

  7. Schulze-Osthoff K et al (1990) In situ detection of basic fibroblast growth factor by highly specific antibodies. Am J Pathol 137(1):85–92

    CAS  PubMed  Google Scholar 

  8. Pakala R, Watanabe T, Benedict CR (2002) Induction of endothelial cell proliferation by angiogenic factors released by activated monocytes. Cardiovasc Radiat Med 3(2):95–101

    Article  PubMed  Google Scholar 

  9. Polverini PJ et al (1977) Activated macrophages induce vascular proliferation. Nature 269(5631):804–806

    Article  CAS  PubMed  Google Scholar 

  10. Sunderkotter C et al (1991) Macrophage-derived angiogenesis factors. Pharmacol Ther 51(2):195–216

    Article  CAS  PubMed  Google Scholar 

  11. Gratchev A et al (2006) Mphi1 and Mphi2 can be re-polarized by Th2 or Th1 cytokines, respectively, and respond to exogenous danger signals. Immunobiology 211(6–8):473–486

    Article  CAS  PubMed  Google Scholar 

  12. Stout RD et al (2005) Macrophages sequentially change their functional phenotype in response to changes in microenvironmental influences. J Immunol 175(1):342–349

    CAS  PubMed  Google Scholar 

  13. Wolfs IM, Donners MM, de Winther MP (2011) Differentiation factors and cytokines in the atherosclerotic plaque micro-environment as a trigger for macrophage polarisation. Thromb Haemost 106(5):763–771

    Article  CAS  PubMed  Google Scholar 

  14. Gordon S (2003) Alternative activation of macrophages. Nat Rev Immunol 3(1):23–35

    Article  CAS  PubMed  Google Scholar 

  15. Mantovani A et al (2004) The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 25(12):677–686

    Article  CAS  PubMed  Google Scholar 

  16. Mantovani A et al (2002) Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol 23(11):549–555

    Article  CAS  PubMed  Google Scholar 

  17. Mosser DM, Edwards JP (2008) Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8(12):958–969

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Kodelja V et al (1997) Differences in angiogenic potential of classically vs alternatively activated macrophages. Immunobiology 197(5):478–493

    Article  CAS  PubMed  Google Scholar 

  19. Lin EY et al (2006) Macrophages regulate the angiogenic switch in a mouse model of breast cancer. Cancer Res 66(23):11238–11246

    Article  CAS  PubMed  Google Scholar 

  20. Pollard JW (2004) Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer 4(1):71–78

    Article  CAS  PubMed  Google Scholar 

  21. Murdoch C et al (2008) The role of myeloid cells in the promotion of tumour angiogenesis. Nat Rev Cancer 8(8):618–631

    Article  CAS  PubMed  Google Scholar 

  22. Sica A et al (2008) Macrophage polarization in tumour progression. Semin Cancer Biol 18(5):349–355

    Article  CAS  PubMed  Google Scholar 

  23. De Palma M et al (2003) Targeting exogenous genes to tumor angiogenesis by transplantation of genetically modified hematopoietic stem cells. Nat Med 9(6):789–795

    Article  PubMed  Google Scholar 

  24. Kanters E et al (2004) Hematopoietic NF-kappaB1 deficiency results in small atherosclerotic lesions with an inflammatory phenotype. Blood 103(3):934–940

    Article  CAS  PubMed  Google Scholar 

  25. Dirkx AE et al (2006) Monocyte/macrophage infiltration in tumors: modulators of angiogenesis. J Leukoc Biol 80(6):1183–1196

    Article  CAS  PubMed  Google Scholar 

  26. Zhang X et al (2006) Receptor specificity of the fibroblast growth factor family. The complete mammalian FGF family. J Biol Chem 281(23):15694–15700

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. He H et al (2012) Endothelial cells provide an instructive niche for the differentiation and functional polarization of M2-like macrophages. Blood 120(15):3152–3162

    Article  CAS  PubMed  Google Scholar 

  28. Murakami M et al (2011) FGF-dependent regulation of VEGF receptor 2 expression in mice. J Clin Invest 121(7):2668–2678

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. Jih YJ et al (2001) Distinct regulation of genes by bFGF and VEGF-A in endothelial cells. Angiogenesis 4(4):313–321

    Article  CAS  PubMed  Google Scholar 

  30. Taraboletti G et al (2002) Shedding of the matrix metalloproteinases MMP-2, MMP-9, and MT1-MMP as membrane vesicle-associated components by endothelial cells. Am J Pathol 160(2):673–680

    Article  CAS  PubMed  Google Scholar 

  31. Presta M et al (2005) Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis. Cytokine Growth Factor Rev 16(2):159–178

    Article  CAS  PubMed  Google Scholar 

  32. Anghelina M et al (2004) Monocytes and macrophages form branched cell columns in matrigel: implications for a role in neovascularization. Stem Cells Dev 13(6):665–676

    Article  CAS  PubMed  Google Scholar 

  33. Fantin A et al (2010) Tissue macrophages act as cellular chaperones for vascular anastomosis downstream of VEGF-mediated endothelial tip cell induction. Blood 116(5):829–840

    Article  CAS  PubMed  Google Scholar 

  34. Lamagna C, Aurrand-Lions M, Imhof BA (2006) Dual role of macrophages in tumor growth and angiogenesis. J Leukoc Biol 80(4):705–713

    Article  CAS  PubMed  Google Scholar 

  35. Rolny C et al (2011) HRG inhibits tumor growth and metastasis by inducing macrophage polarization and vessel normalization through downregulation of PlGF. Cancer Cell 19(1):31–44

    Article  CAS  PubMed  Google Scholar 

  36. Lewis C, Murdoch C (2005) Macrophage responses to hypoxia: implications for tumor progression and anti-cancer therapies. Am J Pathol 167(3):627–635

    Article  CAS  PubMed  Google Scholar 

  37. Leek RD et al (1996) Association of macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma. Cancer Res 56(20):4625–4629

    CAS  PubMed  Google Scholar 

  38. Coffelt SB et al (2010) Angiopoietin-2 regulates gene expression in TIE2-expressing monocytes and augments their inherent proangiogenic functions. Cancer Res 70(13):5270–5280

    Article  CAS  PubMed  Google Scholar 

  39. Porta C et al (2009) Cellular and molecular pathways linking inflammation and cancer. Immunobiology 214(9–10):761–777

    Article  CAS  PubMed  Google Scholar 

  40. De Palma M et al (2005) Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors. Cancer Cell 8(3):211–226

    Article  PubMed  Google Scholar 

  41. Ribatti D, Levi-Schaffer F, Kovanen PT (2008) Inflammatory angiogenesis in atherogenesis–a double-edged sword. Ann Med 40(8):606–621

    Article  CAS  PubMed  Google Scholar 

  42. Khallou-Laschet J et al (2010) Macrophage plasticity in experimental atherosclerosis. PLoS ONE 5(1):e8852

    Article  PubMed Central  PubMed  Google Scholar 

  43. Stout RD, Suttles J (2004) Functional plasticity of macrophages: reversible adaptation to changing microenvironments. J Leukoc Biol 76(3):509–513

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

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.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Molecular Genetics, Cardiovascular Research Institute Maastricht (CARIM), Maastricht, The Netherlands

    Nadine Jetten, Marion J. Gijbels & Marjo M. P. C. Donners

  2. Department of Physiology, Cardiovascular Research Institute Maastricht (CARIM), Maastricht, The Netherlands

    Sanne Verbruggen & Mark J. Post

  3. Department of Medical Biochemistry, Academic Medical Center (AMC), Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands

    Menno P. J. De Winther

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  1. Nadine Jetten
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  2. Sanne Verbruggen
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  3. Marion J. Gijbels
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  5. Menno P. J. De Winther
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  6. Marjo M. P. C. Donners
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Corresponding author

Correspondence to Menno P. J. De Winther.

Electronic supplementary material

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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)

10456_2013_9381_MOESM2_ESM.tif

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)

10456_2013_9381_MOESM3_ESM.tif

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

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