Figure 2.

Angiogenic potential of neutrophils and M2 macrophages depends on production of proMMP-9. (A and B) Neutrophils and macrophages (Mϕs), derived from WT and Mmp9-KO BM, were analyzed for their angiogenic potential in vivo as intact (3 × 10⁴) cells (A) or their secretates (B). Mature BMD-Mϕs (M0 phenotype) were polarized toward the M1 or M2 phenotype. In (B), recombinant TIMP-1 was added to WT neutrophil releasate and M2 Mϕ CM. Pooled data from three (A) and seven (B) independent experiments, each employing from four to six embryos grafted with four to six onplants per variant, are presented. Data are means ± SEM. *P < .05, **P < .005, ***P < .0001. (C) Western blot analysis of MMP-9 (top) and TIMP-1 (bottom) produced by BM neutrophils and BMD-Mϕs was performed in comparison with recombinant proMMP-9 (105 kDa) and TIMP-1 (28 kDa) standards. (D) Zymographic (top) and Western blot analyses of MMP-9 (middle) and TIMP-1 (bottom) secreted by Mϕs were performed in comparison with recombinant proMMP-9 (105 kDa) and TIMP-1 (28 kDa) standards. Mature M0 Mϕs were polarized toward the M1 or M2 phenotype. A portion of M2 Mϕs was then reversed toward the M1 phenotype. (E) Analysis of arginase-1 and iNOS expression in M1, M2, and M2→M1 repolarized Mϕs. (D) Angiogenic potential of mature, polarized, and repolarized Mϕs was determined in vivo as described in A. One of two independent experiments employing from five to seven embryos, each grafted with four to six onplants per variant, is presented. Bars are means ± SEM. *P < .05.