Myeloid angiogenic cells act as alternative M2 macrophages and modulate angiogenesis through interleukin-8

Abstract

Endothelial progenitor cells (EPCs) promote angiogenesis, and clinical trials have shown such cell therapy to be feasible for treating ischemic disease. However, clinical outcomes have been contradictory owing to the diverse range of EPC types used. We recently characterized two EPC subtypes, and identified outgrowth endothelial cells as the only EPC type with true progenitor and endothelial characteristics. By contrast, myeloid angiogenic cells (MACs) were shown to be monocytic cells without endothelial characteristics despite being widely described as "EPCs." In the current study we demonstrated that although MACs do not become endothelial cells or directly incorporate into a microvascular network, they can significantly induce endothelial tube formation in vitro and vascular repair in vivo. MAC-derived interleukin-8 (IL-8) was identified as a key paracrine factor, and blockade of IL-8 but not vascular endothelial growth factor (VEGF) prevented MAC-induced angiogenesis. Extracellular IL-8 transactivates VEGFR2 and induces phosphorylation of extracellular signal-regulated kinases. Further transcriptomic and immunophenotypic analysis indicates that MACs represent alternative activated M2 macrophages. Our findings demonstrate an unequivocal role for MACs in angiogenesis, which is linked to paracrine release of cytokines such as IL-8. We also show, for the first time, the true identity of these cells as alternative M2 macrophages with proangiogenic, antiinflammatory and pro-tissue-repair properties.

Figure 1

MACs induce retinal microvascular network formation. (A) Green-labeled RMECs formed tubes in Matrigel. (B) Red-labeled MACs promoted RMEC tube formation. Scale bars: 500 μm. (C) Higher magnification of coculture showing red-labeled MACs did not incorporate into the vascular network. Scale bar: 200 μm. (D) Quantification of tube length. (D) Quantification of tube area. (E) Addition of MAC-CM significantly increased RMEC tube formation as measured by tube length when compared with nonconditioned media. Data expressed as mean ± SEM. ***P < 0.001 versus control; *P < 0.05 versus control.

Figure 2

MACs promote vascular repair in the ischaemic retina. (A) Injected MACs labeled with red Qdots did not incorporate into the resident vascular network labeled in green with isolectin. Scale bars: 100 μm. (B) Injected human MACs retained a myeloid phenotype as demonstrated by expression of human-specific CD68 and typical amoeboid morphology. Scale bars: 75 μm. (C) Representative flat mounted retinas injected with vehicle (Control) or MACs, respectively. Isolectin staining in green identifies retinal vasculature, and avascular regions are outlined in yellow. Insets show ischaemic areas in white. (D) Quantification of avascular areas and ratio of avascular/total retinal area shows that injection of MACs into the eye significantly induced vascular repair compared with injection of vehicle injected. *P < 0.05 versus control.

Figure 3

Transcriptome analysis revealed that MACs represent angiogenic M2-activated macrophages. (A) Heat maps of typical M1, M2 markers and proangiogenic genes demonstrated that the MAC gene signature is highly enriched for M2 markers, whereas expression of M1 macrophage markers was weak/low. Proangiogenic genes were also highly expressed. (B) Gene expression profiles of MACs were normalized to monocytes. Proangiogenic genes (green) and M2 markers (purple) were upregulated and M1 markers (red) were downregulated compared with housekeeping genes (blue).

Figure 4

Angiogenic cytokines produced by MACs. (A) A human angiogenesis proteome profiler array was used to identify soluble factors released by cells to the medium. RMECs secrete minimal amounts of ET-1, and medium from a MAC and RMEC coculture system showed significant expression of proangiogenic proteins such as MMP-9, IL-8, and MCP-1. Three pairs of positive controls are included in the top corners and bottom left corner. A pair of negative control spots in the bottom right corner indicates very low background levels. (B) IL-8 proteome array assessment of conditioned media of RMECs, MACs and RMECs + MACs. (C) Quantification of proteome profiler shown in (B) by densitometry (O. D., optical density); N = 6. (D) ELISA quantification of IL-8 in conditioned media of RMECs, MACs and RMECs + MACs. N = 4. Data in (C) and (D) expressed as mean ± SEM. ***P < 0.001 versus RMECs; *P < 0.05 versus RMECs; ns, not significant.

Figure 5

The MAC proangiogenic paracrine effect is dependent on secreted IL-8, but independent of extracellular VEGF. Using a PKH kit, we prelabeled RMECs in green and MACs in red before culturing them in the 3D Matrigel Tubulogenesis Assay. Representative images are shown of RMECs alone forming tubes (A); cocultures of RMECs and MACs (B); addition of IL-8 neutralizing antibody to a coculture system (C); and addition of neutralizing VEGF antibody to cocultures (D). Scale bars for A–D: 250 μm. (E) Quantification of tube areas in response to treatments. Anti–IL-8 significantly decreased RMEC tube formation; however, neutralizing VEGF had no significant effect on RMEC tubulogenesis. Data are expressed as mean ± SEM. **P < 0.01 versus control; *P < 0.05 versus control; ns: not significant.

Figure 6

Vascular repair induced by MAC delivery was blocked by CXCR2 inhibitor SB225002. Flat mounted retina microvasculature was stained green with isolectin, and avascular areas (insets) are outlined in white. A representative retina per group is shown: (A) ischemic retina at P12; (B) vehicle-injected retina at P15; (C) MAC-injected retina at P15; (D) MAC-injected retina at P15 in mouse that received SB225005 intraperitoneal injections; (E) vehicle-injected retina at P15 in mouse that received SB225005 intraperitoneal injections; (F) quantification of avascular areas shows that SB225005 significantly inhibited MAC proangiogenic effects. N = 7; ***P < 0.001; ns: not significant.

Figure 7

IL-8 transactivates VEGFR2 and induces ERK phosphorylation in endothelial cells. (A) HMEC-1 cells were treated with 100 ng/mL IL-8 for 15 min, followed by immunoblot analysis using a phospho-VEGFR2 antibody. IL-8 increased VEGFR2 phosphorylation levels compared with controls; 100 ng/mL VEGF treatment was used as a positive control. (B) RMECs were also treated with 100 ng/mL IL-8 for 15 min, and immunoblot analysis for phospho-ERKs was performed. IL-8 induced ERK phosphorylation in RMECs; this effect was completely abolished by the CXCR2 inhibitor SB225002.