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. 2016 Oct;19(4):513-24.
doi: 10.1007/s10456-016-9523-8. Epub 2016 Jul 27.

Regulation of lymphangiogenesis in the diaphragm by macrophages and VEGFR-3 signaling

Alexandra M Ochsenbein  1 , Sinem Karaman  1 , Steven T Proulx  1 , Rhea Goldmann  1 , Jyothi Chittazhathu  1 , Athanasia Dasargyri  1 , Chloé Chong  1 , Jean-Christophe Leroux  1 , E Richard Stanley  2 , Michael Detmar  3
Affiliations

Regulation of lymphangiogenesis in the diaphragm by macrophages and VEGFR-3 signaling

Alexandra M Ochsenbein et al. Angiogenesis. 2016 Oct.

Abstract

Lymphatic vessels play important roles in fluid drainage and in immune responses, as well as in pathological processes including cancer progression and inflammation. While the molecular regulation of the earliest lymphatic vessel differentiation and development has been investigated in much detail, less is known about the control and timing of lymphatic vessel maturation in different organs, which often occurs postnatally. We investigated the time course of lymphatic vessel development on the pleural side of the diaphragmatic muscle in mice, the so-called submesothelial initial diaphragmatic lymphatic plexus. We found that this lymphatic network develops largely after birth and that it can serve as a reliable and easily quantifiable model to study physiological lymphangiogenesis in vivo. Lymphangiogenic growth in this tissue was highly dependent on vascular endothelial growth factor receptor (VEGFR)-3 signaling, whereas VEGFR-1 and -2 signaling was dispensable. During diaphragm development, macrophages appeared first in a linearly arranged pattern, followed by ingrowth of lymphatic vessels along these patterned lines. Surprisingly, ablation of macrophages in colony-stimulating factor-1 receptor (Csf1r)-deficient mice and by treatment with a CSF-1R-blocking antibody did not inhibit the general lymphatic vessel development in the diaphragm but specifically promoted branch formation of lymphatic sprouts. In agreement with these findings, incubation of cultured lymphatic endothelial cells with conditioned medium from P7 diaphragmatic macrophages significantly reduced LEC sprouting. These results indicate that the postnatal diaphragm provides a suitable model for studies of physiological lymphangiogenic growth and maturation, and for the identification of modulators of lymphatic vessel growth.

Keywords: Development; Lymphatic vessels; Macrophages; VEGFR-3.

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Conflict of interest statement

None.

Figures

Fig. 1
Fig. 1
Lymphatic vessels on the pleural side of the diaphragmatic muscle develop and mature postnatally. Whole mounts of C57BL/6 mice show that on the pleural side of the diaphragmatic muscle, LYVE-1-positive vessel structures (green) co-stain for Prox-1 (red) at P7 (a–c) and for CD31 (red) at P5 (d–f). Wide-field images of LYVE-1 whole mounts demonstrate few LYVE-1-positive vessel structures at E16.5 and E18.5 and an expansion of the vessel network from P0 to P7 (g). $ mark unspecific staining of liver tissue. Dotted lines mark the area of the diaphragm that is used for whole-mount imaging and quantifications (g). Merged confocal images of lateral diaphragm segments stained for LYVE-1 show the expansion of the vessel plexus starting from P0 to 6 W (h). Stars mark the central tendon region. High-magnification confocal images allow the visualization of diaphragmatic lymphatic vessel sprouts at P7 (i arrows). Prox-1 (cyan), α-SMA (red) and CD31 (green) whole-mount stainings show valves (arrows) and SMC coverage of lymphatic vessels, located close to the thorax wall (j). CD31 (red) and LYVE-1 (cyan) diaphragm whole mounts of a 6-week-old Prox-1 GFP mouse show LYVE-1 down-regulation on diaphragmatic lymphatic vessels located close to the thorax wall (K-N). Scale bars a–f 100 µm, g and h 1 mm, i 50 µm, j 100 µm, k–n 50 µm. (Color figure online)
Fig. 2
Fig. 2
Diaphragmatic lymphatic vessel growth is VEGFR-3 dependent. Segments of diaphragm whole mounts stained for LYVE-1 of P7 WT (a) and K14-VEGFR-3-Fc transgenic (TG) littermates (b). Quantification of diaphragmatic lymphatic vessel (LV) development of WT and K14-VEGFR-3-Fc transgenic (TG) pups (c–f). Dots represent mean values per mouse, and lines indicate the group means. As only 2 out of 6 diaphragm segments of TG pups had detectable vessels, no statistical analysis was performed for the parameters average LV branch length (e) and LV diameter (f). Segments of LYVE-1-stained pleural diaphragm whole mounts of P5 pups treated with IgG control (g) or antibodies blocking VEGFR-1 (mF1) (h), VEGFR-2 (DC101) (i) or VEGFR-3 (mF4) (j) in utero at E16.5 and E18.5. Quantification of diaphragmatic lymphatic vessel development of VEGFR-blocking antibody-treated pups (k–n). Dots represent mean values per mouse, and lines indicate the group means. Scale bars 1 mm. *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 3
Fig. 3
VEGFR-3 blockade leads to similar lymphatic vessel phenotypes in the tail dermis and on the pleural diaphragm side. LYVE-1 whole mounts of tail dermis of P7 WT (a) and K14-VEGFR-3-Fc transgenic littermates (b). LYVE-1 whole mounts of tail dermis of P5 pups treated with IgG (c) or blocking antibodies against VEGFR-1 (mF1) (d), VEGFR-2 (DC101) (e), or VEGFR-3 (mF4) (f) in utero at E16.5 and E18.5. VEGFR-3 blockade leads in both approaches to an almost complete inhibition of the lymphangiogenic process, whereas VEGFR-1 and -2 blockades have no obvious effect. Scale bar 100 µm
Fig. 4
Fig. 4
Macrophages are closely aligned with lymphatic vessels and their sprouting tips and develop before lymphatic vessels. Diaphragm whole mounts stained for LYVE-1 (red), CD206 (cyan) and F4/80 (green) at P8 show macrophages aligned in parallel to lymphatic vessels (a). P7 diaphragm whole mounts show that LYVE-1 (green)- and CD206 (cyan)-positive macrophages have close contact with the sprouting tip of LYVE-1-positive lymphatic vessels (b, arrows), but they are not positive for Prox-1 (red) (c). E14.5 diaphragm whole mounts stained for CD206 (red), LYVE-1 (cyan) and CD31 (green) show the presence of CD206- and LYVE-1-positive macrophages but no LYVE-1-positive vessel structures (d). At E14.5, the few Prox-1+ lymphatic endothelial cells (cyan) do not co-stain for CD68 (green) (e, arrow). Scale bars a 50 µm, b and c 50 µm, d 100 µm, e 20 µm. (Color figure online)
Fig. 5
Fig. 5
Colony-stimulating factor-1 receptor-positive macrophages negatively regulate lymphatic vessel branching on the pleural side of the diaphragm. P6 diaphragm whole mounts show CSF-1R and LYVE-1 double-positive macrophages in proximity to LYVE-1-positive lymphatic vessels (a). Compared to WT littermates (b), CD206 (cyan)- and CD68 (red)-stained whole mounts of diaphragms of P7 Csf1r−/− pups show depletion of macrophages (c). Segments of pleural diaphragm LYVE-1 whole mounts of P7 WT (d) or Csf1r−/− pups (e). CD68-stained whole mounts of IgG (f)- or AFS98 (g)-treated pups show depletion of macrophages on the pleural side of the diaphragmatic muscle by AFS98 treatment. Quantification of the CD68-positive area per visual field in the diaphragm (h). Dots represent mean values per mouse, and lines indicate the group means. Quantification of diaphragmatic lymphatic vessel development of Csf1r−/− pups shows a significant increase in branches and lymphatic loops compared to the WT controls (k–o). Dots represent mean values per mouse, and lines indicate the group means. Segments of pleural diaphragm LYVE-1 whole mounts of P7 IgG (i)- or AFS98-treated pups (j). Quantification of diaphragmatic lymphatic vessel development of P7 AFS98-treated pups shows a significant increase in branches, lymphatic loops and a significant decrease in average branch length compared to the IgG controls (p–t). Dots represent mean values per mouse, and lines indicate the group means. Quantifications of LEC sprouts per bead show that isolated P7 diaphragmatic macrophage-conditioned medium significantly decreased VEGF-A/FGF-induced LEC sprouting (u). Dots represent mean values per well, and lines indicate the group means. Scale bars a 50 µm, b and c 50 µm, d and e 1 mm, f and g 50 µm, i and j 1 mm. *p < 0.05; **p < 0.01; ***p < 0.001. (Color figure online)
Fig. 6
Fig. 6
Macrophage depletion does not impair lymphatic vessel drainage function. Stereomicroscope image showing fluorescent microspheres within mediastinal lymph nodes (a, arrows) in exposed thorax 1 h after i.p. injection of microspheres into P8 pup. T thymus, H heart, R rib cage. FACS analysis in the FSC/SSC (b) and FITC/FSC (c) channels. FACS analysis of green–yellow fluorescent microspheres in PBS in mediastinal lymph nodes of uninjected pups (d), and in non-draining inguinal lymph nodes (e) and in draining mediastinal lymph nodes (f) 1 h after i.p. injection of microspheres into WT P8 pups. Quantification by FACS showed that there was no difference in drainage of fluorescent microspheres to pooled mediastinal lymph nodes 1 h after i.p. injection of microspheres into rat IgG- or AFS98-treated P7 pups (g)
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