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Endothelin-1 Stimulates Lymphatic Endothelial Cells and Lymphatic Vessels to Grow and Invade

¹ Molecular Pathology and ² Immunology Laboratories, Regina Elena Cancer Institute and ³ Molecular Biology and Pathology Institute, National Research Council, Rome, Italy; and ⁴ Microbiology Section, Department of Experimental Medicine, University of Brescia, Brescia, Italy

Requests for reprints: Anna Bagnato, Laboratory of Molecular Pathology, Regina Elena Cancer Institute, Via delle Messi d'Oro 156, 00158 Rome, Italy. Phone: 39-06-52662565; Fax: 39-06-52662600; E-mail: bagnato{at}ifo.it.

Key Words: endothelin B receptor • endothelin-1 • lymphatic endothelial cells • vascular endothelial growth factor • hypoxia-inducible factor-1

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_BR). 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_BR. 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)-1 expression similarly to hypoxia. Moreover, HIF-1 silencing by siRNA desensitizes VEGF-C and VEGF-A production in response to ET-1 or hypoxia, implicating HIF-1/VEGF as downstream signaling molecules of ET-1 axis. Double immunofluorescence analysis of human lymph nodes reveals that lymphatic vessels express ET_BR together with the lymphatic marker podoplanin. Furthermore, a Matrigel plug assay shows that ET-1 promotes the outgrowth of lymphatic vessels in vivo. ET_BR blockade with the specific antagonist, BQ788, inhibits in vitro and in vivo ET-1–induced effects, demonstrating that ET-1 through ET_BR directly regulates lymphatic vessel formation and by interacting with the HIF-1–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_BR and related signaling cascade may be therapeutically exploited in a variety of diseases including cancer. [Cancer Res 2009;69(6):2669–76]