Fig. 5: Distinct requirements for VEGFR2 and VEGFR3 in VEGF-C-induced lymphangiogenesis.

a Experimental scheme and ligand-receptor interactions for assessing VEGF-C-induced lymphangiogenic response after genetic deletion of Vegfr2, Vegfr3 or Pik3ca in the mature lymphatic vasculature. Two weeks after induction mice received an intradermal (i.d.) injection of AAV-VEGF-C into the ear skin and were analyzed 2 weeks later. VEGF-CΔNΔC, fully processed mature form of VEGF-C capable of binding VEGFR2. VEGF-C-induced lymphangiogenic response in mice lacking lymphatic endothelial Vegfr3 (b), Pik3ca (c, quantified in d) or Vegfr2 (e), and their respective littermate controls. Note loss of lymphatic hyperplasia in Vegfr3-deleted (b, 1 mg of Tam) and Pik3ca-deleted (c, 3 ×1 mg of Tam) mice and reduced sprouting in Vegfr2-deleted mice (e, 3 × 1 mg of Tam). Data in (d) represent mean vessel area (n = 3–4 mice per genotype as indicated) ± s.d., ***p < 0.001 and ****p < 0.0001, Two-sided Mann-Whitney U test. Images in (b, e) represent data from 3 independent experiments. Flow cytometry analysis of LEC proliferation after VEGF-C stimulation, showing reduced proliferation (KI67⁺) and expansion of the total LEC population in Vegfr3-deleted mice (f), while Vegfr2-deleted LECs show similar proliferation and only a modest reduction in expansion of LEC population (g) compared to controls. Data represent mean % of LECs (n = 4-9 mice per genotype as indicated) ± s.d. Dotted lines indicate %KI67⁺ LECs and proportion of LECs of all ECs in untreated wild type (+/+) mice (f, g). **p < 0.01, ***p < 0.001 and ****p < 0.0001; Two-sided Mann Whitney U test. Early time-point analysis of lymphatic capillary morphology and LEC proliferation (KI67 + PROX1 + , yellow arrows) in AAV-VEGF-C-treated mice (h) and in Pik3ca^H1047R;Vegfr3-CreER^T2 mice following topical application of 4-OHT to activate PI3Kα (i). 3-5 mice were analysed for each time point, and the number of vessel ends analyzed is indicated. Scale bar: 250 µm (b, c, e), 50 µm (h, i).