Inhibition of lymphangiogenesis in vitro and in vivo by the multikinase inhibitor nintedanib

PURPOSE: To investigate the feasibility of nintedanib, a novel triple angiokinase inhibitor, for inhibiting lymphatic endothelial cell (LEC)-induced lymphangiogenesis in vitro and inflammatory corneal lymphangiogenesis in vivo. MATERIALS AND METHODS: Methylthiazolyldiphenyl-tetrazolium bromide (MTT) test, transwell system, and tube-formation assay were used to evaluate the effects of nintedanib on the proliferation, migration, and tube formation of LECs stimulated by vascular endothelial growth factor-C (VEGF-C), basic fibroblast growth factor (bFGF), or platelet-derived growth factor-BB (PDGF-BB). The murine model of suture-induced corneal neovascularization was used to assess the anti-hemangiogenic and anti-lymphangiogenic effects of nintedanib via systemic and topical applications. Corneal flatmounts were stained with lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and CD31, and the areas of involved blood and lymph vessels were analyzed morphometrically. Corneal cryosections were stained with F4/80 to evaluate inflammatory cell recruitment. RESULTS: We observed a significant enhanced effect of LEC proliferation, migration, and tube formation with the administration of VEGF-C, PDGF-BB, and bFGF, respectively, which was diminished by nintedanib. Both topical and systemic applications of nintedanib inhibited suture-induced hemangiogenesis and lymphangiogenesis in the murine cornea. A reduction in F4/80(+) cell infiltration was observed at day 14 after corneal suture for both systemic and topical applications of nintedanib. In comparison with controls, 61% of F4/80(+) cell recruitment was inhibited via the systemic application of nintedanib, while 49% of F4/80(+) cell recruitment was inhibited with the topical application of nintedanib. CONCLUSION: Nintedanib was shown to inhibit in vitro lymphangiogenesis stimulated by VEGF-C, bFGF, and PDGF-BB. Applied topically or systemically, it effectively inhibited corneal hemangiogenesis and lymphangiogenesis, accompanied by reduced inflammatory cell recruitment, which represents a new promising treatment for graft rejection after penetrating keratoplasty.