Abstract 17097: M2 Macrophage Polarization Promotes Neovascularization in vivo

Abstract

Following myocardial infarction (MI), resident and recruited macrophages remove necrotic and apoptotic cells, secrete cytokines, and modulate angiogenesis at the infarct site. In response to microenvironmental stimuli, macrophages adopt either pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes; however, the role of macrophage polarity in tissue neovascularization is unclear. We hypothesized that macrophage polarity with regard to activation state plays a critical role in neovascularization during wound healing. Peritoneal macrophages were isolated from adult C57Bl/6 mice (n = 7) using thioglycollate-elicitation, and polarization was induced to either M1, using LPS 1 μg/ml and IFN-γ 4 ng/ml, or M2, using IL-4 20 ng/ml or IL-10 20 ng/ml. Successful macrophage polarization was confirmed by immunofluorescence for M1- and M2-specific surface markers. In syngeneic mice, Matrigel plugs (0.6 ml/plug) were injected subcutaneously in vivo, either alone or with M1 or M2 macrophages (~20,000 cells/plug) or supernatant derived from M1 or M2 cultures (0.3 ml/plug) and plug neovascularization was assessed after 10 days. In addition, we also performed in vitro scrape wound healing assays using cultured mouse cardiac endothelial cells (MCECs) and human umbilical vein endothelial cells (HUVECs) and tube formation assays with HUVECs. Supernatant derived from M2 macrophages induced faster wound healing over 16 h in cultured MCECs or HUVECs, and increased HUVEC tube formation and tube length. However, in vitro wound closure and tube formation was delayed by supernatant derived from M1 cells (n = 5). In vivo neovascularization in the Matrigel plug was quantified morphologically and by flow cytometry. The presence of M2 macrophages, or supernatant derived from M2 macrophages, resulted in increased neovessel length and number, with recruitment of significantly greater numbers of CD31+ cells to the Matrigel plug, as compared with M1 or inactivated macrophages and their associate culture supernatant (p < 0.05, n = 6). We conclude that M2 macrophage polarization promotes angiogenesis in vivo via paracrine effects on vessel recruitment. Strategies to induce M2 polarization in vivo may augment wound healing after MI.