Blood vessel control of macrophage maturation promotes arteriogenesis in ischemia

Abstract

Ischemia causes an inflammatory response that is intended to restore perfusion and homeostasis yet often aggravates damage. Here we show, using conditional genetic deletion strategies together with adoptive cell transfer experiments in a mouse model of hind limb ischemia, that blood vessels control macrophage differentiation and maturation from recruited monocytes via Notch signaling, which in turn promotes arteriogenesis and tissue repair. Macrophage maturation is controlled by Notch ligand Dll1 expressed in vascular endothelial cells of arteries and requires macrophage canonical Notch signaling via Rbpj, which simultaneously suppresses an inflammatory macrophage fate. Conversely, conditional mutant mice lacking Dll1 or Rbpj show proliferation and transient accumulation of inflammatory macrophages, which antagonizes arteriogenesis and tissue repair. Furthermore, the effects of Notch are sufficient to generate mature macrophages from monocytes ex vivo that display a stable anti-inflammatory phenotype when challenged with pro-inflammatory stimuli. Thus, angiocrine Notch signaling fosters macrophage maturation during ischemia.Molecular mechanisms of macrophage-mediated regulation of artery growth in response to ischemia are poorly understood. Here the authors show that vascular endothelium controls macrophage maturation and differentiation via Notch signaling, which in turn promotes arteriogenesis and ischemic tissue recovery.

Fig. 1

Origin of macrophages in ischemia. a Representative flow cytometric analysis of ischemic tibialis anterior muscle of Cx3cr1 ^GFP/+ mice. Granulocytes (green), Ly6C^hi monocytes (blue) and macrophages (black), n = 3 mice/group. b Adoptive cell transfer at d1 after HLI and cell fate tracking of CD45.2⁺CD11b⁺GFP⁺Ly6C^hi monocytes transferred into CD45.1⁺ recipients. Representative flow cytometry of recipient muscle, replicated at least three times. c Treatment with CCR2 blocking antibody MC-21 (α) or isotype control c for indicated time intervals (r × d) after HLI and analysis of cell populations in peripheral blood (PB) and muscle (M) by flow cytometry. n = 5/7/7 mice/group, error bars represent s.e.m. ***p < 0.001, **p < 0.01, *p < 0.05 *indicates comparison between treatment with MC-21 (α) vs isotype control c by one way ANOVA with Bonferroni multiple comparison test. d Quantitative RT-PCR analysis of cell populations of Cx3cr1 ^GFP/+ mice sorted from d3 ischemic muscle relative to bone marrow (BM) Ly6C^hi monocytes. n = 3 independent experiments, error bars represent s.e.m. ***p < 0.001, **p < 0.01, *p < 0.05, ^#Comparison between muscle and BM Ly6C^hi cells, *Comparison between muscle Ly6C^hi and muscle macrophages (MF), one way ANOVA with Bonferroni multiple comparison post-test

Fig. 2

Ischemia triggers induction of Notch ligand Dll1 in arteries. a Quantitative RT-PCR analysis of cell populations of Cx3cr1 ^GFP/+ mice sorted from d3 ischemic muscle relative to bone marrow (BM) Ly6C^hi monocytes. n = 3 independent experiments, error bars represent s.e.m. ***p < 0.001, **p < 0.01, *p < 0.05 ^#Comparison between muscle and BM Ly6C^hi cells, *Comparison between muscle Ly6C^hi and macrophages (MF), One way ANOVA with Bonferroni multiple comparison post-test b Quantitative RT-PCR analysis of ischemic muscle at indicated time points d. n = 3/4/4/4 mice/group, error bars represent s.e.m., one way ANOVA with Dunnett's Multiple Comparison test. c β-galactosidase activity (blue) and CD45 immunohistochemistry (red) in ischemic muscle from Dll1 ^+/lacZ reporter mice. Scale bars=50 µm. d Representative immunostaining and confocal microscopy of collateral arteries in Cx3cr1 ^GFP/+ mice after HLI. Phase contrast image (GFP) × 5, scale bar=200 µm. Confocal images with anti-SMA × 40, CD31 × 40 and × 63. Nuclear counterstain (DAPI, blue). Scale bar=20 µm

Fig. 3

DLL1 instruct macrophage maturation in vitro. a Flow cytometry of Ly6C^hi monocytes from Cx3cr1 ^GFP/+ mice cultured for 3d on IgG-Fc (con) or DLL1-Fc chimeric proteins, in the presence of 10 ng/ml CSF1. Representative of n = 5 independent experiments. b Quantitative RT-PCR analysis at d3 after culture with CSF1, CSF2. n = 2 independent experiments performed in duplicates, error bars represent s.e.m. **p < 0.01, *p < 0.05 by one way ANOVA with Bonferroni multiple comparison post-test. c Representative analysis of proliferation by 6 h BrdU incorporation at d3. n = 3 independent experiments. d GFP⁺ cell numbers in culture relative to input number. n = 3 independent experiments performed in duplicates, error bars represent s.e.m. **p < 0.01 by two-way ANOVA and Bonferroni multiple comparison post-test. e Quantitative RT-PCR analysis normalized to gene expression levels of input Ly6C^hi monocytes (d0). n = 4 independent experiments performed in duplicates, error bars represent s.e.m. ***p < 0.001, **p < 0.01, *p < 0.05 by two way ANOVA with Bonferroni multiple comparison post-test. f BrdU incorporation in CD11b⁺ cells at day 7 after 100 ng/ml CSF1 pulse. n = 3 independent experiments, error bars represent s.e.m. ***p < 0.001 by Student’s unpaired t-test. g Representative Annexin V/7-AAD staining gated on CD11b⁺ macrophages, n = 3 independent experiments, error bars represent s.e.m, **p < 0.01, Student’s unpaired t test. h Representative flow cytometry of phagocytosis (1 h) of PE-labeled dextran beads at indicated temperature, d3 of culture. n = 3 independent experiments, error bars represent s.e.m, *p < 0.05, Student’s unpaired t test. i Multiplex measurement of cytokine concentration in culture supernatant after stimulation with 100 ng/ml LPS (6 h) or combination of 20 ng/ml IFNγ (18 h) and LPS (6 h). n = 4 independent experiments, error bars represent s.e.m. **p < 0.01, *p < 0.05, Student’s paired t test. j, k Quantitative RT-PCR analysis after stimulation with IFNγ/LPS normalized to input monocyte levels. Comparison of cells stimulated j at d3 or d6, k with or without re-stimulation with CSF1 (100 ng/ml) at d6. n = 4 independent experiments in duplicates, error bars represent s.e.m. *p < 0.05 by Student’s paired t-test

Fig. 4

Endothelial Dll1 instructs macrophage maturation in vitro. a Quantitative RT-PCR analysis of p3 primary human aortic EC (HAEC, A) or coronary microvascular (MV) EC. n = 3 independent samples in duplicates/group, error bars represent s.e.m., **p < 0.01, Student’s unpaired t-test. b Representative flow cytometry from d3 culture of human CD14⁺ monocytes in the presence of 10 ng/ml M-CSF or HAEC (AEC). n = 4 independent experiments. c, d HAEC were transfected with anti-DLL1 siRNA (Si-DLL1) or scambled control (Scr) before co-culture with CD14⁺ monocytes. c. Quantitative RT-PCR analysis of CD11b⁺ isolated from co-culture normalized to expression levels of input monocytes, n = 3 independent experiments, error bars represent s.e.m. **p < 0.01, *p < 0.05, Student’s paired t-test. d BrdU incorporation (6 h) in CD11b⁺ cells from co-culture, n = 3 independent experiments performed with duplicate measurements, error bars represent s.e.m. *p < 0.05, Student’s unpaired t-test. e Protein concentration of CSF1 in co-culture supernatants (A: arterial EC, MV: microvascular EC) or macrophage (MF) supernatants by ELISA, nd: not detectable. n = 3 independent experiments performed in triplicates, error bars represent s.e.m. ***p < 0.001 one-way ANOVA with Bonferroni post-test. f Number of CD11b⁺ macrophages recovered after co-culture of CD14⁺ monocytes with HAEC treated with isotype (α-Con) and CSF1 neutralizing (α-CSF1) antibody. n = 2 independent experiments performed in triplicates, error bars represent s.e.m. p < 0.0001, Student’s unpaired t-test g, h Effect of HAEC (A) or MVEC (MV) on CD14⁺ monocytes. g Representative flow cytometry and profile of gated population after d3 of co-culture. n = 3 independent experiments. h Transcriptional profile of CD11b⁺ cells isolated after 3d of culture with EC (A vs MV) or CSF1, normalized to expression levels of input monocytes (d0). n = 3 independent experiments, error bars represent s.e.m. ***p < 0.001, **p < 0.01, *p < 0.05, one way ANOVA with Bonferroni multiple comparison test

Fig. 5

Endothelial Dll1 regulates macrophage maturation and arteriogenesis. a Analysis of cell populations by flow cytometry in ischemic muscle of induced endothelial Dll1 mutant mice (Dll1 ^iΔEC) and control mice. n = 8 mice/group, error bars represent s.e.m. *p < 0.05 by two way ANOVA with Bonferroni multiple comparison test. b Gene expression analysis of macrophages sorted from d3 ischemic muscle. n = 3 independent experiments, error bars represent s.e.m. **p < 0.01, *p < 0.05, Student’s unpaired t-test. c–f Rescue of endothelial Dll1 deficiency after HLI by transfer of ischemic macrophages. On d1 after HLI, indicated groups received intramuscular limb injections of 5 × 10e5 macrophages isolated from d3 ischemic muscle (MF) or Ly6C^hi monocytes isolated from spleen (Mo) of wt mice, or received control treatment. c Foot perfusion by laser Doppler perfusion measurement immediately after induction of HLI (0) and during follow up. n = 5/7/7/7 mice/group, error bars represent s.e.m. ***p < 0.001 by two way ANOVA and Bonferroni post test. d Quantification of inner circumference and wall area, and representative H&E stained images of collateral arteries d14. Scale bar=50 μm. n = 7 mice/group, error bars represent s.e.m. ***p < 0.001 vs control by one way ANOVA and Dunnett’s multiple comparison test. e Representative SMA-stained immunofluorescence of muscle sections, Scale bar 100 μm (left) and quantification of arterial branches (SMA⁺) per section d14, n = 5 mice/group, error bars represent s.e.m. ***p < 0.001 vs control by one way ANOVA and Dunnett’s multiple comparison test. f Representative H&E stained muscle sections. Scale bar=50 μm (left) and quantification of ghost cells and regenerating muscle fibers (right) at d14, n = 5 mice/group, error bars represent s.e.m. ***p < 0.001 vs control by one way ANOVA and Dunnett’s multiple comparison test

Fig. 6

Canonical Notch signaling regulates macrophage maturation in vivo. a Analysis of cell populations by flow cytometry in ischemic muscle of Rbpj conditional mutant mice (Rbpj ^ΔM) and control mice. n = 7/9 mice/group, error bars represent s.e.m., *p < 0.05 by two way ANOVA with Bonferroni post test. b Representative flow cytometry of 6 h in vivo BrdU incorporation in macrophages from ischemic muscle at d3. n = 3 independent experiments c Representative flow cytometry of macrophages (CD11b⁺, F4/80⁺) from ischemic muscle at d3. n = 4 independent experiments d Gene expression analysis of macrophages sorted from d3 ischemic muscle. n = 3 independent experiments, error bars represent s.e.m. ***p < 0.001, **p < 0.01, *p < 0.05, unpaired Student’s t-test. e Representative cell death analysis in macrophages (CD11b⁺, F4/80⁺) from ischemic muscle. n = 4 independent experiments. f Gene expression analysis of isolated Ly6C^hi monocytes from wild type or Rbpj ^∆M mice (0) and derived macrophages cultured on DLL1 for indicated time. n = 3 independent experiments measured in duplicates, error bars represent s.e.m. ***p < 0.001, **p < 0.01, two-way ANOVA and Bonferroni post-test. g Representative BrdU incorporation of d7 macrophages on DLL1. n = 4 independent experiments

Fig. 7

Macrophage Notch signaling regulates arteriogenesis in vivo. a Foot perfusion by laser Doppler perfusion measurement after induction of HLI (0) and during follow up. n = 8/10 mice/group, error bars represent s.e.m. *p < 0.05, ***p < 0.001 vs control by two way ANOVA and Bonferroni post-test. b Representative LDI images of collateral circulation at d3. c Representative H&E images of collateral arteries and quantification of inner circumference, n = 4/6 (ctl), 9/8 (d3), 7/7 (d14) mice/group, error bars represent s.e.m. *p < 0.05, ***p < 0.001 vs control by two way ANOVA and Bonferroni post-test. Scale bar=100 μm. d Overview (×10), magnification (×63) of representative H&E stained muscle sections and quantification of necrosis, n = 5 mice/group, error bars represent s.e.m. ***p < 0.001 vs control by Student’s unpaired t-test. Scale bar=100 μm. e Representative H&E stained muscle sections, quantification of ghost cells and regenerating muscle fibers, n = 6/7 mice/group, error bars represent s.e.m. **p < 0.001, Student’s unpaired t-test. Scale bar=100 μm. f Representative images of Masson-trichrome staining and quantification of fibrosis in muscle sections at d14. n = 6/5 mice/group, error bars represent s.e.m. **p < 0.01, Student’s unpaired t-test. Scale bar=100 μm. g–i Adoptive transfer of monocytes on d1 after HLI in Rbpj ^ΔM mice. Intravenous injections of PBS (Rbpj ^ΔM) or 3 × 10e5 BM Ly6C^hi monocytes from wild type (+Con) or Rbpj ^ΔM mice (+Rbpj ^ΔM). g Foot perfusion by laser Doppler imaging after induction of HLI (0) and during follow up. n = 6/5/5 (d0), 7/5/5 (d3), 6/5/5 (d7), 7/5/5 (d14) mice/group, error bars represent s.e.m. ***p < 0.001, **p < 0.01, *p < 0.05, *comparison between Rbpj ^ΔM and +Con group, # Comparison between +Con and +Rbpj ^ΔM groups, Two way ANOVA and Bonferroni post test. h Representative H&E stained images of collateral arteries at d14 and quantification of collateral circumference, n = 7/5/5 mice/group, error bars represent s.e.m. *p < 0.05, ***p < 0.001, one way ANOVA and Bonferroni post-test. Scale bar=100 μm. i Representative images of Masson-trichrome staining and quantification of fibrosis in muscle sections at d14, n = 7/5/5 mice/group, error bars represent s.e.m. *p < 0.05, ***p < 0.001, one way ANOVA and Bonferroni post-test. Scale bar=100 μm