TIMP-1–deficient status determines high angiogenic capacity of proMMP-9 produced by human M2-polarized macrophages. (A) Complexing of neutrophil and M2 macrophage proMMP-9 with TIMP-1. Purified TIMP-free neutrophil proMMP-9 and TIMP-deficient proMMP-9 produced by M2 macrophages was incubated with fivefold molar excess of TIMP-1 and repurified by affinity chromatography on gelatin Sepharose to remove unbound TIMP-1. The original and resulting preparations were analyzed by western blotting for 92-kDa MMP-9 and 28-kDa TIMP-1, confirming efficient complexing of TIMP-1 to proMMP-9. (B) Encumbrance by TIMP-1 abrogates high angiogenesis-inducing capacity of proMMP-9. Preparations of original and TIMP-1–complexed proMMP-9 from neutrophils and M2 macrophages were incorporated into collagen mixtures to provide 3 ng of proMMP-9 per onplant. The levels of angiogenesis were analyzed 3 days later in comparison with PBS control. The data are from a representative experiment performed using from 4 to 6 embryos, each grafted with 6 collagen onplants, per condition. The data are presented as fold changes (means ± SEM) in the levels of angiogenesis induced by different proMMP-9 preparations compared with PBS control (1.0). ***P < .0001. (C) Downregulation of TIMP-1 expression by siRNA treatment. After 7-day differentiation of monocytes into mature M0 macrophages and M0 macrophage polarization into M1 and M2 phenotypes, the cells were transfected with control or TIMP-1 siRNA constructs. CM from siRNA-treated mature M0 and polarized M1 and M2 macrophages was analyzed by western blotting for levels of proMMP-9 and TIMP-1. Note that TIMP-1 siRNA treatment did not affect the levels of proMMP-9 production, but significantly downregulated TIMP-1 expression in M0 and M1 macrophages. (D) Downregulation of TIMP-1 in the TIMP-1–expressing M0 and M2 macrophages induces their angiogenic capacity. Low levels of TIMP-1 expression and production correlates with high levels of angiogenesis induced by mature and polarized macrophages. Mature M0 and polarized M1 and M2 macrophages were treated with control or TIMP-1 siRNA and incorporated into collagen mixtures to provide 1 × 10⁴ cells per collagen onplant. Negative control contained no cells (NC). The data are presented as fold changes (means ± SEM) in the levels of angiogenesis compared with NC control (1.0). Note that significant downregulation of TIMP-1 in M0 and M1 macrophages results in a significant increase of their angiogenic capacity. **P < .01.