Figure 7.

Synergistic interaction of human podoplanin-positive monocytes (PPMs) and platelets led to lymphatic neovascularization and facilitated wound healing in nude mice. (a) Gross appearances of the wounds at the back of nude mice injected with the different cells at the indicated time points. Scale bar = 2 mm. (b) Diagram of the kinetics of wound closure in each group. Four mice were analyzed at each time point (*P < 0.05). (c–g) Mice that had received surgery for wound to back or ear skin were injected with the different cells, and the tissues were harvested at 7 days for immunohistochemistry. (c) In the confocal images of back skin from nude mice of indicated group, the LYVE-1-positive lymphatic vessels were more abundant after transplantation of platelets and PPMs than monocytes alone, which were reversed by blocking antibody against podoplanin. Scale bar = 100 µm. Blue fluorescence indicates DAPI. (d) Bar graph representing the quantification of lymphatic vessel number in the back skin, which was determined by scoring LYVE-1-positive vessels (**P < 0.01; n = 4 per experiment). (e) Whole-mount immunostaining of ear skin stained with antibodies against LYVE-1. Scale bar = 100 µm. Blue fluorescence indicates DAPI. Red dots denote branching points. LYVE-1-positive lymphatic vessels arborized more abundantly after transplantation of platelet and PPMs than monocytes alone, which was reversed by blocking antibody against podoplanin. (f) Bar graph representing the number of lymphatic vessels–branching points (**P < 0.01; n = 4 per experiment). (g) Whole-mount immunostaining of ear skin wound treated with PPMs and platelets. About half of these transplanted human cells were stained as red (HLA-ABC) and coexpressed LYVE-1 (green), suggesting that these cells transdifferentiated into lymphatic vessels in mice. Scale bar = 10 µm. Blue fluorescence indicates DAPI. (h) Confocal images of back skin from female nude mice treated with PPMs from human male volunteers. Transplanted human cells were stained with SRY (red) and hLYVE-1 (green), and mouse lymphatic vessel was stained with mLYVE-1 (white). Blue fluorescence indicates DAPI. DAPI, 4′,6-diamidino-2-phenylindole; HLA-ABC, human leukocyte antigen-ABC; hLYVE-1, human lymphatic vessel endothelial hyaluronan receptor-1; LYVE-1, lymphatic vessel endothelial hyaluronan receptor-1; mLYVE-1, mouse lymphatic vessel endothelial hyaluronan receptor-1; PBS, phosphate-buffered saline; PDPN Ab, podoplanin-blocking antibody; SRY, sex-determining region Y.