Figure 2: TNF-α induces TNFR1-dependent corneal lymphangiogenesis.

(a) Confocal images of TNF-α-, VEGF-C- and vehicle-induced LYVE-1⁺ lymphangiogenesis (green) and CD31⁺ angiogenesis (red) in mouse corneas. Red arrowheads indicate lymphatic vessels; white arrowheads indicate blood vessels. Scale bar, 200 μm. Quantification of corneal CD31⁺ blood angiogenic and LYVE-1⁺ lymphangiogenic vessels (n=4 mice per group). (b) High magnification of LYVE-1⁺ (green) lymphangiogenic vessels that were co-stained with TNFR1 (red). Arrows point LYVE-1 and TNFR1 double-positive structures. Scale bar, 10 μm. (c) Confocal images of LYVE-1⁺ lymphangiogenesis (green) and CD31⁺ angiogenesis (red) in mouse corneas. TNF-α-induced angiogenesis and lymphangiogenesis in Tnfr1^−/− mice and in an anti-VEGFR3 neutralizing antibody-treated wild type (wt) mice were shown. Vehicle-treated mice served as a control. Arrowheads in upper panels indicate lymphatic vessels; arrows in lower panels point to filopodia tips sprouting from angiogenic lymphatic vessels. The diameters of lymphatic vessels are indicated. Scale bar, 200 μm (upper panels); 20 μm (lower panels). (d) Quantification of TNF-α-induced corneal CD31⁺ angiogenesis and LYVE-1⁺ lymphangiogenesis in Tnfr1^−/− mice, in VEGFR3 blockade-treated wt mice and in vehicle-treated wt mice (n=4 mice per group). *P<0.05; **P<0.01; ***P<0.001; NS, not significant. All error bars represent s.e.m. All P values were analysed according to Student’s t-test.