Figure 5. Systemic delivery of VEGF-C markedly increases lymphatic drainage and mobilization of inflammatory cells from the inflamed colon to DLNs.
(A) 10 μg of Evans blue dye was injected into the colonic mucosa of healthy (UN, n = 4/time point), Ad-hVEGF-C– (n = 5/time point), and mF431C1-treated mice (n = 5/time point) undergoing 2 cycles of DSS treatment. Mice treated with DSS alone (n = 5/time point) were used as a control group. Evans blue was then extracted 16 hours after the dye injection from distal colons of comparable weight. Graph shows the total dye remaining in the colon expressed in μg. Data represent the mean per group ± SEM. Black dashed lines represent the 2 DSS cycles. (B–D) GFP+ peritoneal inflammatory cells (~106 cells) were injected into the rectal mucosa of the inflamed and noninflamed colons of healthy (UN, n = 5) and DSS-treated mice (n = 5 per experimental group, at day 5 after the second DSS cycle) and IL-10 WT (n = 4) and IL-10–KO mice (n = 4 per group at day 21 after the first Ad-hVEGF-C administration). Twelve hours later, the DLNs were sampled and sectioned for histologic and FACS analysis. (C) Upper panels: Representative frozen sections of DLNs from the indicated experimental groups coimmunostained for LYVE1 (red) and DAPI (blue) and merged. Lower panels: Representative FACS from the indicated groups. The number in each FACS histogram represents the percentage of GFP+ cells. Scale bar: 50 μm. (D) GFP+ inflammatory cells in the entire frozen section were quantified and are presented as AU. Error bars represent the mean per group ± SEM. *P < 0.05; **P < 0.001.