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

• Published in: Cancer research (Chicago, Ill.) Ill.), 2009-03, Vol.69 (6), p.2669-2676
• Main Authors: SPINELLA, Francesca, GARRAFA, Emirena, DI CASTRO, Valeriana, ROSANO, Laura, RITA NICOTRA, Maria, CARUSO, Arnaldo, GIORGIO NATALI, Pier, BAGNATO, Anna
• Format: Article
• Language: English
• Subjects: Antineoplastic agents; Biological and medical sciences; Cell Growth Processes - physiology; Endothelial Cells - metabolism; Endothelial Cells - pathology; Endothelin-1 - biosynthesis; Endothelin-1 - metabolism; Humans; Hypoxia-Inducible Factor 1, alpha Subunit - metabolism; Lymphangiogenesis; Medical sciences; Pharmacology. Drug treatments; Receptor, Endothelin B - biosynthesis; Signal Transduction; Tumors; Vascular Endothelial Growth Factor A - biosynthesis; Vascular Endothelial Growth Factor C - biosynthesis; Vascular Endothelial Growth Factor Receptor-3 - biosynthesis
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/0008-5472.CAN-08-1879

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.