Lymphangiogenesis-independent resolution of experimental edema

Abstract

Vascular endothelial growth factor (VEGF)-C is necessary for lymphangiogenesis, and excess VEGF-C has been shown to be ameliorative for edema produced by lymphatic obstruction in experimental models. However, it has recently been shown that edema can resolve in the mouse tail even in the complete absence of capillary lymphangiogenesis when distal lymph fluid crosses the regenerating wound site interstitially. This finding has raised questions about the action of VEGF-C/VEGF receptor (VEGFR) signaling during the resolution of experimental edema. Here, the roles of VEGFR-2 and VEGFR-3 signaling in edema resolution were explored. It was found that edema resolved following neutralization of either VEGFR-2 or VEGFR-3 in the mouse tail skin, which inhibited lymphangiogenesis. Neutralization of either VEGFR-2 or VEGFR-3 reduced angiogenesis at the site of obstruction at day 10 (9.2 +/- 1.2% and 11.5 +/- 1.0% blood capillary coverage, respectively) relative to controls (14.3 +/- 1.5% blood capillary coverage). Combined VEGFR-2/-3 neutralization more strongly inhibited angiogenesis (6.9 +/- 1.5% blood capillary coverage), leading to a reduced wound repair of the lymphatic obstruction and extended edema in the tail skin. In contrast, improved tissue repair of the obstruction site increased edema resolution. Macrophages in the swollen tissue were excluded as contributing factors in the VEGFR-dependent extended edema. These results support a role for VEGFR-2/-3-combined signaling in the resolution of experimental edema that is lymphangiogenesis independent.

Fig. 1.

Edema resolution may be dependent on combined VEGF receptor (VEGFR)-2/-3 signaling. Edema in the mouse tail skin was induced over a period of 60 days by a 1-mm-wide surgical excision of the skin that was left unprotected. Injury sites at days 20 and 30 for control, VEGFR-3 neutralization, VEGFR-2 neutralization, and combined receptor neutralization conditions are shown from top to bottom, respectively. Scale bar (A, bottom, right) = 5 mm. The evolution of wound closure (B) and tail swelling (C) over the 60-day period for the different conditions is graphically depicted. For the wound closure data, wound lengths are normalized to the initial 1-mm-long wound. For the swelling data, tail diameters are normalized to the average initial (preswelling) diameter of the tails within each group. n = 10 mice per group. *Statistical significance.

Fig. 2.

Edema resolution in the mouse tail skin is enhanced by accelerated tissue repair at the site of obstruction. Edema in the mouse tail skin was induced by a 1-mm-wide surgical excision of the skin that was left unprotected or protected with a silicone cuff. Images are shown at 0, 2, 5, 10, 15, and 25 days postsurgery (A). Scale bar (A, bottom, right) = 5 mm. B: tail diameters in the normal and protected tissue regeneration conditions. *P < 0.001, differences at day 15 and day 25 between these groups were highly significant. Tail diameters were normalized to their average initial (day 0) value. n = 10 mice.

Fig. 3.

Tissue repair promotes fluid drainage across the obstruction site. Edema in the mouse tail was induced by a 1-mm-wide surgical incision, and microlymphangiographies were conducted at 60 days in tails that were left uncovered (A) and treated with anti-VEGFR-3 (B), anti-VEGFR-2 (C), or combined anti-VEGFR-2/-3 blocking antibodies (D) or covered with a cuff that enhanced wound repair (E). Distal to proximal direction is right to left in all images. Bar (in E) = 1 mm. It was seen that fluid tracer diffused slowly across the wound site in A–D and was transported within regenerated lymphatics (identified by unique hexagonal architecture) in E. Regenerated lymphatic function was assessed at day 60 by measuring fluid tracer clearance across the obstruction via microlymphangiography in the covered and uncovered tails (F). *Statistical significance. A–E: panels consist of multiple images at different locations of the mouse tail that were collected under the fluorescence microscope and then assembled in Photoshop to form a complete representation of the respective tails.

Fig. 4.

Tissue repair improves regeneration of lymphatic function. Sections (10 μm thin) of the uncovered (A) and covered (B) tail skin were immunostained against podoplanin (distal to proximal direction shown right to left), showing fluid tracer (red) overlap with interstitium (blue, cell nuclei) and lymphatic capillaries (green). Distal to proximal direction is right to left in the images. Several hyperplastic and poorly functioning lymphatics are identified by yellow arrowheads in A, and several lymphatics colocalizing with tracer are identified by yellow arrows in B. Bar (in H) = 1 mm. Regenerated region is located between the white bars for each image. C and D: enlargements of the 1-mm-long regenerating regions from images in A and B, respectively. n = 10 mice per group. A–D: panels consist of multiple images at different locations of the tissue section that were collected under the fluorescence microscope and then assembled in Photoshop to form a complete representation of the respective tissue.

Fig. 5.

Dependence of macrophage infiltration and angiogenesis during experimental edema on VEGFR signaling. Edema in the mouse tail skin was induced by a 1-mm-wide surgical excision of the skin that was placed 10 mm from the tail base and left unprotected. Shown are fluorescence images of the F4/80 macrophage marker (A–E) and CD31 blood endothelial cell marker (F–J) (immune-detected antigen in each image is shown in red). Macrophages were detected in tissue sections of normal mouse tail skin (A), in edematous mouse tail skin (B), and in edematous mouse tail skin treated with anti-VEGFR-3 neutralizing antibodies (C), anti-VEGFR-2 neutralizing antibodies (D), and combined receptor signaling neutralization (E). In B–E, which depict macrophages in the swollen skin, the skin epidermis is located at the top of each image. Scale bar (in E) = 0.5 mm. Blood endothelial cells were detected in tissue sections of normal mouse tail skin (F) or the distal wound margin in edematous mouse tail skin treated with isotype matched control antibody (G) or in edematous mouse tail skin treated with anti-VEGFR neutralizing antibodies (H), anti-VEGFR-2 neutralizing antibodies (I), and combined receptor signaling neutralizing antibodies (J). Wound margin is shown on the right side in G–J, indicated by yellow arrowheads. Blue color in A–J is 4′,6-diamino-2-phenylindole (DAPI)-labeled cell nuclei. Scale bar (in J) = 0.5 mm. Measurement of blood capillary percent coverage for the different treatments within 500 μm of the wound edge (K) demonstrated inhibition of angiogenesis in the regenerating tissue by the receptor blocking antibodies. n = 5 mice per group. *Significant difference relative to the control condition. **Significant difference relative to VEGFR-3 blocking. A–J: panels consist of multiple images at different locations of the tissue section that were collected under the fluorescence microscope and then assembled in Photoshop to form a complete representation of the respective tissue.