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. 2020 Dec;190(12):2355–2375. doi: 10.1016/j.ajpath.2020.08.018

Figure 4.

Figure 4

Lung lymphatics associated with lymphocyte influx and dependence on vascular endothelial growth factor receptor (Vegfr) 3 signaling and Vegfc expression. A: Lung lymphatics (green) near B-cell follicles (red) in lymphoid tissue near pulmonary vein (bleomycin, 14 days). B: Lymphatics near B and T cells in follicles (bleomycin, 14 days). C: Lung lymphatic packed with B and T cells (bleomycin, 56 days). D: Lymphatics near high endothelial venules (HEVs; MECA-79 staining) in lung hilum (bleomycin, 7 days). E and F: Low-magnification panoramas of C57BL/6 lung lymphatics 14 days after bleomycin administration without (E) or with concurrent Vegfr3 blockade by mF4-31C1 (F). G: Abundance of lung lymphatics (area density) at baseline (blue dots) or 14 days after bleomycin treatment with concurrent treatment (red symbols) with IgG, mF4-31C1 (anti-Vegfr3), DC101 (anti-Vegfr2), or both antibodies. Symbols or dots represent individual mice. H–K: Increased expression of Vegfc mRNA (quantitative RT-PCR) and protein [enzyme-linked immunosorbent assay (ELISA)] 14 days after bleomycin administration (black dots) without changes in expression of Vegfd or Vegfa (quantitative RT-PCR) over baseline group (blue dots). L: Positive control staining for Vegfc to validate antibody. Triple-transgenic prospero homeobox 1-enhanced green fluorescent protein (Prox1-EGFP)/CCSP/VEGF-C mouse given doxycycline for 7 days to induce Vegfc in epithelial club cells (arrows) in bronchus. M: Staining for Vegfc, macrophages [allograft inflammatory factor 1/ionized calcium binding adapter molecule 1 (Aif1/Iba1)], and lymphatics in 14-day bleomycin lung. Macrophages (arrows) stain for Vegfc. n = 4 to 10 mice per group (G). ∗P < 0.05 versus baseline (t-test). Scale bars: 1 mm (A, E, and F); 100 μm (B and D); 50 μm (C and M); 20 μm (L). Pecam1, platelet endothelial cell adhesion molecule.