Tissue macrophages act as cellular chaperones for vascular anastomosis downstream of VEGF-mediated endothelial tip cell induction

Abstract

Blood vessel networks expand in a 2-step process that begins with vessel sprouting and is followed by vessel anastomosis. Vessel sprouting is induced by chemotactic gradients of the vascular endothelial growth factor (VEGF), which stimulates tip cell protrusion. Yet it is not known which factors promote the fusion of neighboring tip cells to add new circuits to the existing vessel network. By combining the analysis of mouse mutants defective in macrophage development or VEGF signaling with live imaging in zebrafish, we now show that macrophages promote tip cell fusion downstream of VEGF-mediated tip cell induction. Macrophages therefore play a hitherto unidentified and unexpected role as vascular fusion cells. Moreover, we show that there are striking molecular similarities between the pro-angiogenic tissue macrophages essential for vascular development and those that promote the angiogenic switch in cancer, including the expression of the cell-surface proteins TIE2 and NRP1. Our findings suggest that tissue macrophages are a target for antiangiogenic therapies, but that they could equally well be exploited to stimulate tissue vascularization in ischemic disease.

Figures

Figure 1

Spatiotemporal relationship of tissue macrophages and blood vessels during angiogenesis in the developing hindbrain. Eighty-micron transverse sections (A-C) and whole mounts (D-H) of 10.5-, 11.5-, and 12.5-dpc wild-type hindbrains, labeled for IB4 (red) and F4/80 (green). Solid white arrows indicate macrophages positive for both markers; clear arrows, macrophages positive for IB4 only; curved arrows, macrophages embracing vascular intersections; arrowheads indicate endothelial tip cells. (G-H) Higher magnifications of the SVP illustrate the interaction of tip cells (arrowheads) with macrophages (arrows) and the bridging of neighboring tip cells by a macrophage (wavy arrow in G; note that one of the 2 vessel sprouts emerges from a deeper plane of section); panel H shows a higher magnification of the boxed area in panel E. Scale bars: panels A through F, 100 mm; panels G and H, 25 mm. V indicates ventricular brain surface; p, pial brain surface; rv, radial vessels; hb, hindbrain; SVP, subventricular vascular plexus; and PNP, perineural vascular plexus. (I) Quantitation of macrophages (Mϕ, green) and vascular intersections in the SVP (red) between 10.5 and 12.5 dpc; n ≥ 15. Error bars represent SD of the mean.

Figure 2

Tissue macrophages bridge endothelial tip cells and are present at vascular junctions. Hindbrains from 11.5- and 12.5-dpc wild-type embryos were fluorescently labeled with IB4 (red) and F4/80 (green) to reveal the relationship of endothelial cells (red) and macrophages (double-positive, yellow) in the subventricular zone. (A) A macrophage (arrow) interacts with the filopodia of 2 opposing tip cells (arrowheads) at E11.5. (B) A macrophage embraces a 3-way junction, the product of vessel fusion (curved arrow). (C-D) Snapshots of 3-dimensional models obtained by surface rendering of the confocal z-stacks shown in panels A and B. Panels C′ and D′ show different angles of the models shown in panels C and D, respectively. Scale bar represents 25 μm.

Figure 3

Brain angiogenesis is impaired in the absence of macrophages. Angiogenesis in hindbrains lacking PU.1 (Pu.1−/−) or heparin-binding VEGF isoforms (Vegfa120/120) at 12.5 dpc (A-L) and 11.5 dpc (M-P). (A-G) Whole mount view onto the SVP (A-C) or radial vessels (RV) diving into the brain parenchyma (E-G), visualized by PECAM immunohistochemistry (IHC) in a 0.25-mm2 area. (I-K) Transverse sections (100 μm) of IB4-labeled hindbrains show vascular bridges (white arrowheads) between neighboring radial vessels. (M-O) IB4-positive endothelial tip cells (clear arrowheads) and macrophages (clear arrows) in the whole-mounted SVP. Scale bars represent 100 μm (A-K) and 50 μm (M-O). (D,H,L,P) Quantitation of the number of vascular intersections in the SVP (D; n = 5), the number of radial vessels (H; n = 5), and the number of vascular bridges between radial vessels (L; n = 3). Error bars represent SD of the mean. P values were determined by comparing the measurements obtained for both types of mutants to the control, which contained the measurements obtained for wild-types from both groups.

Figure 4

Macrophages promote SVP angiogenesis independently of VEGF. (A-F) Hindbrains (11.5 dpc) were labeled for IB4 and F4/80; (D-F) higher magnifications of the boxed areas in (A-C). Controls (no Cre; A,D) were compared with littermates with a knockdown of VEGF in neural progenitors (NesCreVegfafl/+; B,E) and stage-matched mutants lacking VEGF164 only (Vegfa120/120; C,F). Scale bar represents 100 μm. (G) Quantitation of the number of vascular intersections and macrophages (Mϕ) in the SZ at 11.5 dpc; n = 3. Error bars represent SD of the mean. The P values were determined by comparing both types of mutants to Vegfafl/+ hindbrains lacking Cre. (H) Quantitation of Vegfa mRNA levels relative to Actb (β-actin) in PU.1-deficient and wild-type littermate hindbrains at 11.5 dpc. The horizontal lines indicate the mean; n = 3.

Figure 5

VEGF-induced vessel sprouting and macrophage-mediated anastomosis act synergistically to promote vascular network formation. (A-F) Morphology of the SVP (IB4-positive, red) and distribution of tissue macrophages (IB4/F4/80-double positive, yellow) in 11.5 dpc hindbrains of the indicated genotypes; arrows in panel D indicate macrophages interacting with endothelial tip cells in Vegfa120/120 mutants; arrowheads in panels D through F denote examples of bulbous vessel ends, typical of Vegfa120/120 mutants. Scale bar represents 100 μm. (G) Quantitation of the number of vascular intersections in the SZ of the indicated genotypes at 11.5 dpc; n > 3. Error bars represent SD of the mean. (H) Schematic illustration of the role of macrophages during hindbrain vascularization.

Figure 6

TEM antigen expression by proangiogenic hindbrain macrophages. (A-B) Wild-type hindbrains (11.5 dpc) were double-labeled for IB4 and NRP1 to visualize the subventricular zone, including the SVP; the boxed areas in panels A, A′, and A′′ are shown in higher magnifications in panels B, B′, and B′′, respectively; yellow indicates colocalization in panels A′′ and B′′. Many NRP1-positive macrophages (arrows in B,B′′) interacted with endothelial tip cells (arrowheads in panel B′′); a NRP1-positive macrophage bridging neighboring tip cells is indicated with a wavy arrow in panels B and B′′. (C-G) Double labeling for NRP1 and the macrophage markers CD11b (CD18/MAC-1), F4/80, or IBA1 (C-E), the vascular endothelial marker PECAM (F), or the neural progenitor marker SOX2 (G) established that NRP1 was expressed in all 3 cell types. Yellow indicates coexpression of NRP1 with macrophage and endothelial markers on the cell surface; in contrast, NRP1 surrounds the SOX2-positive neural progenitor nuclei. The images shown in panels F and G are stacks through the SVP or neural progenitor layer only. (H-I) The specificity of the NRP1 antibody was established by immunolabeling a Nrp1-deficient hindbrain, obtained from a littermate embryo of the wild-type shown in panels A and B. (J-K) Like NRP1, TIE2 (red) colocalized with IB4 (green) on a filopodia-bearing endothelial tip cell (arrowhead) and interacting tissue macrophage (arrow). Scale bars represent 25 μm (except A-A′′, 100 μm).

Figure 7

Retinal angiogenesis is impaired in the absence of tissue macrophages. Double-labeling of transverse sections through a wild-type mouse eye at 11.5 dpc (A) and a wild-type mouse retina at P21 (B) with IB4 and F4/80 (A) or an antibody specific for smooth muscle actin (SMA); cell nuclei in panel B were labeled with DAPI. (C-I) Whole mount labeling of the wild-type retinal vasculature at P4 (C), P9 (D-F) and P21 (G-I). (C) Some macrophages (solid arrows) interact with endothelial tip cells (solid arrowheads) at the vascular front; others embrace emerging vascular bridges (wavy arrows). (D-E) Activated caspase 3 (aC3) is present in regressing vessel segments near arteries (yellow); note capillary narrowing at the junction of the regressing vessel segment with a stable vessel (open arrowheads) and a macrophage at one of the narrow ends (solid arrow); the area boxed in panel D is shown at higher magnification in panel E. (F) Collagen IV and IB4 expression are retained by acellular capillaries (feathered arrow). (G-I) IB4-positive vessel regression profiles (red arrows) and stable intersections (red arrowheads) between nonregressing vessel segments at P21 in the deep plexus of the indicated genotypes. Scale bars represent 50 μm (except in A, 100 μm). (J-K) Quantitation of stable intersections (J) and regression profiles (K) in the deep plexus at P21; n = 3. Error bars represent SD of the mean. P values were determined by comparing the measurements obtained for both types of mutants to the control, which contained the measurements obtained for wild-types from both groups. SP indicates superficial plexus; IP, intermediate plexus; DP, deep plexus; ONL, outer nuclear layer; INL, inner nuclear layer; RGC, retinal ganglion cell layer; OPL, outer plexiform layer; PR, photoreceptor layer; a, artery; and v, vein.

Figure 8

Tissue macrophages interact with endothelial cells at sites of vessel fusion in the developing zebrafish trunk. Series of laser confocal projections extracted from a time-lapse video (supplemental Video 1) showing the interaction of 3 tissue macrophages (red; 1-3) with blood vessels (green); the time elapsed after starting the video at 28 hpf is indicated in hours:minutes in the bottom left of each panel; 3 adjacent intersegmental blood vessels are imaged over a period of 4:16. (A) A macrophage (1) migrates into a region where the tip cell from the caudal sprout of one intersomitic vessel fuses with the tip cell of a rostral sprout of the next intersomitic vessel; arrows indicate the direction of macrophage migration. (B) A macrophage (2) migrates to a site where a dividing endothelial cell in the forming DLAV (clear arrowhead) transiently loses contact with the intersomitic vessel (arrowheads); it remains in this position while the connection is re-established (forked arrowheads); yet another macrophage (3) interacts with filopodial protrusions from opposing tip cells (wavy arrow). Scale bar represents 50 μm.