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. 2020 Jun 2;31(6):1136–1153.e7. doi: 10.1016/j.cmet.2020.05.004

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© 2020 The Author(s)

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Endothelial Lactate Controls Macrophage Polarization and Function upon Muscle Ischemia

(A) Scheme illustrating experimental set-up.

(B) Gene profiling of unstimulated BMDMs (vehicle) or BMDMs stimulated with mECs^wt-CM, mECs^Δpfkfb3-CM.

(C and D) Gene expression analysis of arg1 (C) and mrc1 (D) in BMDMs stimulated with fractionated mECs^wt-CM and mECs^Δpfkfb3-CM, supplemented with lactate (5 mM) where indicated.

(E) Lactate concentration in mECs^wt-CM and mECs^Δpfkfb3-CM.

(F) Representative images of immunostainings of F4/80 (green), CD206 (red), and hoechst (blue) in BMDMs stimulated with mECs^wt-CM, mECs^Δpfkfb3-CM with or without lactate supplementation and quantification of CD206 MFI (n = 3).

(G) OCR upon injection of oligomycin (oligo), FCCP, and rotenone plus antimycin A after mECs^wt-CM, mECs^Δpfkfb3-CM, mECs^wt+lac-CM, and mECs^Δpfkfb3+lac-CM stimulation (n = 4–5).

(H) RNaseq data showing OXPHOS gene expression in muscle macrophages 3 days after HLI (n = 3).

(I and J) Representative images (I) and quantification (J) of proliferating MPCs upon incubation with macrophage-derived CM, measured as percentage of EdU⁺ nuclei (red, EdU⁺; blue, hoechst).

(K and L) MPC fusion analysis: representative images of immunofluorescent DESMIN staining (K) (red, DESMIN; blue, hoechst) and quantification of the number of nuclei per DESMIN⁺ myotube (L).

(M) Vegf gene expression in CD45⁺ cells sorted from pfkfb3^WT and pfkfb3^ΔEC muscle at 3 d. (N) VEGF secretion by BMDMs after stimulation with mECs^wt-CM and mECs^Δpfkfb3-CM with or without lactate supplementation.

Scale bar, 50 μm. Student’s t test (two-tailed, unpaired) in (E) and (M). One-way ANOVA with Tukey’s multiple comparisons test in (B). Two-way ANOVA with Tukey’s multiple comparisons test in (C), (D), (G), (J), (L), and (N) (^∗p < 0.05) as well as in (F) (^∗p < 0.05 versus mECs^wt-CM; ^#p < 0.05 versus ctrl). Each dot represents a single mouse (M) or the average of an independent experiment ([B], [C], [D], [E], [J], [L], and [N]). Bar graphs represent mean ± SEM. See also Figure S5 and Table S1.