Fig. 6. Reprogrammed endogenous glycolysis in macrophages supports their angiogenic activity.

(A to C) Ki-67 and BrdU staining of human RECs. Human RECs were exposed to conditioned medium (CM) from Pfkfb3^WT or Pfkfb3^ΔMϕ BMDMs pretreated with control medium (VCBM) or hypoxic REC-CM (Hx-REC-CM) for 12 hours. n = 6. (D to I) Human RECs were cultured in collagen gel to grow into three-dimensional multicellular spheroids, or on a two-dimensional matrix to form a tube network in the presence or absence of CM from Pfkfb3^WT or Pfkfb3^ΔMϕ BMDMs pretreated with VCBM or Hx-REC-CM. Representative fluorescence photographs of angiogenic tube formation after culturing for 8 hours in matrix (D). Scale bars, 50 μm. Cumulative tube length quantified using the ImageJ software (E) and branch points calculated from five experiments in each case (n = 6) (F). Representative images of spheroidal sprouting after culturing for 24 hours in collagen matrix (G). Scale bars, 100 μm. Morphometric quantification of spheroid sprouting by calculating the number of sprouts per spheroid (H), as well as total sprout length (I). n = 10 per group. n is number of spheroids quantified. a.u., arbitrary units. (J to M) Representative images of sprouting aortic rings (J) and choroids (L) exposed to CM from Pfkfb3^WT or Pfkfb3^ΔMϕ BMDMs pretreated with VCBM or Hx-REC-CM for 12 hours. Sprouting areas were quantified (K and M). (N) Sprouting aortic rings were exposed to CM from Pfkfb3^WT BMDMs, or cocultured with BMDMs from Pfkfb3^WT or Pfkfb3^ΔMϕ in Transwells. Representative images of sprouting aortic rings at day 8 (n = 6 per group). (O) Sprouting areas were quantified. Scale bars, 500 μm. (P) Western blot analysis of Pfkfb3 protein expression in mouse aortic ECs collected from (N). n = 4. Data are means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, by one-way ANOVA followed by Bonferroni test.