title: Endothelin-1 stimulates lymphatic endothelial cells and lymphatic vessels to grow and invade.
abstract: The lymphatic vasculature is essential for tissue fluid homeostasis and cancer metastasis, although the molecular mechanisms involved remain poorly characterized. Endothelin-1 (ET-1) axis plays a crucial role in angiogenesis and tumorigenesis. Here, we first report that ET-1 acts as a lymphangiogenic mediator. We performed in vitro and in vivo studies and show that lymphatic endothelial cells produce ET-1, ET-3, and express the endothelin B receptor (ET(B)R). In these cells, ET-1 promotes proliferation, invasiveness, vascular-like structures formation, and phosphorylation of AKT and p42/44 mitogen-activated protein kinase through ET(B)R. In normoxic conditions, ET-1 is also able to up-regulate the expression of vascular endothelial growth factor (VEGF)-C, VEGF receptor-3, and VEGF-A, and to stimulate hypoxia-inducible factor (HIF)-1alpha expression similarly to hypoxia. Moreover, HIF-1alpha silencing by siRNA desensitizes VEGF-C and VEGF-A production in response to ET-1 or hypoxia, implicating HIF-1alpha/VEGF as downstream signaling molecules of ET-1 axis. Double immunofluorescence analysis of human lymph nodes reveals that lymphatic vessels express ET(B)R together with the lymphatic marker podoplanin. Furthermore, a Matrigel plug assay shows that ET-1 promotes the outgrowth of lymphatic vessels in vivo. ET(B)R blockade with the specific antagonist, BQ788, inhibits in vitro and in vivo ET-1-induced effects, demonstrating that ET-1 through ET(B)R directly regulates lymphatic vessel formation and by interacting with the HIF-1alpha-dependent machinery can amplify the VEGF-mediated lymphatic vascularization. Our results suggest that ET-1 axis is indeed a new player in lymphangiogenesis and that targeting pharmacologically ET(B)R and related signaling cascade may be therapeutically exploited in a variety of diseases including cancer.
authors.affiliation: Molecular Pathology and Immunology Laboratories, Regina Elena Cancer Institute and Molecular Biology and Pathology Institute, National Research Council, Rome, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Spinella
authors.forename: Emirena
authors.initials: E
authors.lastname: Garrafa
authors.forename: Valeriana
authors.initials: V
authors.lastname: Di Castro
authors.forename: Laura
authors.initials: L
authors.lastname: Rosanò
authors.forename: Maria Rita
authors.initials: MR
authors.lastname: Nicotra
authors.forename: Arnaldo
authors.initials: A
authors.lastname: Caruso
authors.forename: Pier Giorgio
authors.initials: PG
authors.lastname: Natali
authors.forename: Anna
authors.initials: A
authors.lastname: Bagnato
mesh_terms: D048708:Cell Growth Processes / Q000502:physiology; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology; D019332:Endothelin-1 / Q000096:biosynthesis / Q000378:metabolism*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D042583:Lymphangiogenesis; D044023:Receptor, Endothelin B / Q000096:biosynthesis; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis; D042582:Vascular Endothelial Growth Factor C / Q000096:biosynthesis; D040321:Vascular Endothelial Growth Factor Receptor-3 / Q000096:biosynthesis
chemical_list: D019332:Endothelin-1; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D044023:Receptor, Endothelin B; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042582:Vascular Endothelial Growth Factor C; D040321:Vascular Endothelial Growth Factor Receptor-3