Ectopic expression of PDGF-B in lymphatic vessels and phosphorylation of PDGFR-β in mural cells leads to aberrant mural cell recruitment during pulmonary fibrosis. (A) Quantitative analysis of the PDGFR-β expression in isolated lymphatic endothelial cells of bleomycin-treated (Bleo) and healthy control mice at day 28 (controls n = 15; Bleo n = 14). Immunostaining of PDGF-B (B) or PDGFR-β (C) and lymphatic vessels (VEGFR3) on lung tissue of fibrotic and control mice at day 28. Scale bars equal 100 μm. (D) Westernblot of phospho–PDGFR-β and total PDGFR-β of whole lung lysates at day 28 after treatment with bleomycin or PBS, respectively (controls n = 5; Bleo n = 13). (E) Quantitative analysis of the ratio of phospho–PDGFR-β to total PDGFR-β at day 28 after treatment with bleomycin or PBS (controls n = 5, Bleo n = 13). (F) Double-immunodetection of phospho–PDGFR-β and lymphatic vessels (VEGFR3) on healthy and fibrotic murine lung tissue at day 28. Scale bars equal 100 μm. Quantitative analysis of Lyve-1–positive lymphatic vessels (G), VEGFR3-positive lymphatic vessels (H) or Prox1-positive lymphatic vessels (I) covered with SMA-positive mural cells at day 28 after treatment with bleomycin, bleomycin + AG-1296 (Bleo + AG-1296; 10 mg/kg body weight twice a week intraperitoneally; 0.5 ng, 5 times per week intraperitoneally, starting at day 14 after first bleomycin injection) or in healthy control lungs of PBS-treated mice (controls n = 15; Bleo n = 29, Bleo + AG-1296, n = 13). Error bars show SEM.