Notch1 controls macrophage recruitment and Notch signaling is activated at sites of endothelial cell anastomosis during retinal angiogenesis in mice - PubMed (original) (raw)

Notch1 controls macrophage recruitment and Notch signaling is activated at sites of endothelial cell anastomosis during retinal angiogenesis in mice

Hasina H Outtz et al. Blood. 2011.

Abstract

Notch is a critical regulator of angiogenesis, vascular differentiation, and vascular integrity. We investigated whether Notch signaling affects macrophage function during retinal angiogenesis in mice. Retinal macrophage recruitment and localization in mice with myeloid-specific loss of Notch1 was altered, as these macrophages failed to localize at the leading edge of the vascular plexus and at vascular branchpoints. Furthermore, these retinas were characterized by elongated endothelial cell sprouts that failed to anastomose with neighboring sprouts. Using Notch reporter mice, we demonstrate that retinal macrophages localize between Dll4-positive tip cells and at vascular branchpoints, and that these macrophages had activated Notch signaling. Taken together, these data demonstrate that Notch signaling in macrophages is important for their localization and interaction with endothelial cells during sprouting angiogenesis.

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Figures

Figure 1

Notch1 in macrophages is important for macrophage localization during retinal angiogenesis. Whole mount P5 retinas were stained by immunohistochemistry for F4/80 or isolectin-B4 to visualize macrophages or endothelial cells, respectively. (A) Macrophages (red) in control retinas were found in close proximity to endothelial cells (green) and at vascular branchpoints (arrows). (B) F4/80 staining of image shown in panel A. Macrophages in control retinas were densely localized to the vascular front, at the leading edge of vessel migration and anastomosis. (C) Macrophages (red) in retinas from Notch1+/− mice were often not associated with vascular branchpoints (indicated with stars). (D) F4/80 staining of image shown in panel C. Macrophages in Notch1+/− retinas were scattered at the vascular front where vessel anastomosis occurs. (E-G) Retinal angiogenesis in mice with myeloid-specific reduction of Notch1 was characterized by an increased frequency of elongated sprouts that failed to anastomose with neighboring sprouts. For quantification of elongated sprouts, 10× images of retinas were used to assess the frequency of sprouts longer than 100 μm, normalized to the total number of sprouts in that field. Sprouts were defined as endothelial protrusions from the vascular front that did not branch. Results were averaged for each genotype. Error bars represent SEM of n = 4 control, n = 6 LysMCre; Notch1 flox/+, and n = 4 LysMCre;Notch1flox/flox retinas. (H-L) Decreased macrophages at the retinal vascular front in mice with myeloid-specific loss of Notch1. For quantification, paneled 30× images of the entire retina were taken. To determine F4/80 staining density (shown in red), the number of red pixels within the outer 20% of the retina (indicated by white line in panels H and I, outline of vascular plexus shown in white) was counted and compared with the total area covered by vasculature within that region. The leading edge was defined as the distal 20% of vasculature measured from the center of the optic nerve to the edge of the vascular front. Error bars represent SEM of n = 4 control and n = 4 LysMCre;Notch1flox/flox retinas. *P ≤ .05. (K-L) Macrophages in control retinas were found at the vascular front and localized to vascular branchpoints (arrowheads), whereas there was decreased macrophage density at the vascular front in retinas from mice with loss of Notch1 in the myeloid lineage (arrows). Images shown at 40× (A-D,K,L), or 10× (E,F,H,I), original magnification. Images are representative of at least 3 independent experiments.

Figure 2

Macrophages with activated Notch signaling localize to anastomosing sprouts and at vascular branchpoints. Whole mount P5 retinas. (A) F4/80 (red) and isolectin-B4 (blue) staining demonstrates localization of macrophages in close proximity to tip cells (arrowhead) and between neighboring sprouts (arrow). (B) Macrophages (green arrow) bridge between neighboring Dll4-postive (red) tip cells (arrowheads). Isolectin-B4 shown in blue. (C-F) Immunohistochemistry of retinas from Notch reporter mice expressing GFP downstream of a Notch-responsive promoter. Anti-GFP staining shown in green indicates activation of Notch signaling. Vessels were visualized with isolectin-B4 (blue). F4/80 staining shown in red. (C) Macrophages (red) with evidence of Notch signaling (arrows) localize between sprouts at the vascular front. (D) Proximally in the vascular plexus, macrophages with evidence of Notch signaling were found in close association with endothelial cells at vascular branchpoints (arrows). (E-F) Higher magnifications of the boxed areas in panels C and D. Arrowheads indicate anastomosing sprouts (E) or vascular branchpoints (F). Macrophages that were not in association with endothelial cells did not have evidence of Notch signal activation (indicated with stars). (G) The majority of TNR-positive macrophages in retinas were localized to vascular branchpoints (87.9% vs 12.07% of TNR-positive macrophages. (H-I) TNR-positive macrophages are overrepresented in the distal 20% of the retinal vasculature. Forty-two percent of all retinal macrophages were located in the distal 20% of the retinal vasculature. Of TNR-positive macrophages, 72.5% were located in the distal 20% of the retina. TNR-positive macrophages circled in white. Error bars represent data from n = 5 retinas from TNR mice. *P ≤ .05. Images in panels A through D shown at 40× original magnification, panel I shown at 4× original magnification. Data are representative of at least 3 independent experiments.

References

1. Checchin D, Sennlaub F, Levavasseur E, Leduc M, Chemtob S. Potential role of microglia in retinal blood vessel formation. Invest Ophthalmol Vis Sci. 2006;47(8):3595–3602. - PubMed
1. Fantin A, Vieira JM, Gestri G, et al. Tissue macrophages act as cellular chaperones for vascular anastomosis downstream of VEGF-mediated endothelial tip cell induction. Blood. 2010;116(5):829–840. - PMC - PubMed
1. Hellstrom M, Phng LK, Hofmann JJ, et al. Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature. 2007;445(7129):776–780. - PubMed
1. Suchting S, Freitas C, le Noble F, et al. The Notch ligand Delta-like 4 negatively regulates endothelial tip cell formation and vessel branching. Proc Natl Acad Sci U S A. 2007;104(9):3225–3230. - PMC - PubMed
1. Swiatek PJ, Lindsell CE, del Amo FF, Weinmaster G, Gridley T. Notch1 is essential for postimplantation development in mice. Genes Dev. 1994;8(6):707–719. - PubMed

Notch1 controls macrophage recruitment and Notch signaling is activated at sites of endothelial cell anastomosis during retinal angiogenesis in mice - PubMed (original) (raw)