Abstract 5280: Activation of sphingosine kinase 1 is necessary for angiopoietin 2-induced hemangiogenesis and lymphangiogenesis

Abstract

Abstract INTRODUCTION: Sphingosine kinase 1 (SphK1) is one of the two isoenzymes that generate the potent bioactive sphingolipid mediator, sphingosine-1-phosphate (S1P). S1P promotes cancer cell survival, proliferation and hemangiogenesis. The angiopoietin2 (Ang2)-Tie2 axis also plays a significant role in hemangiogenesis and lymphangiogenesis. The aim of this study was to investigate the involvement of SphK1 in Ang2-induced hem- and lymphangiogenesis. METHODS: Human umbilical vein endothelial cells (HUVECs) and human lymphatic endothelial cells (HLECs) were treated without or with an isozyme-specific inhibitor of SphK1 (SK1-I) and qPCR, western blot, ELISA, and tube formation assays carried out. We also used a new method we developed to quantify both hem- and lymphangiogenesis in the same sample in vivo by combining Directed In Vivo Angiogenesis Assays (DIVAA) with Fluorescence Activated Cell Sorting (DIVAA/FACS). RESULTS: qPCR revealed that human HLECs like HUVECs express high levels of SphK1 and the S1PR1 receptor. S1P rapidly stimulated xocytosis of Ang2 from HLEC storage granules determined by ELISA. Ang2 activated Tie2 in HLECs as demonstrated by its rapid phosphorylation on Tyr992, a major site responsible for its activation. Similar to Ang2, S1P also stimulated phosphorylation of Tie2, which may be due to secretion of Ang2. S1P is a potent hem- and lymphangiogenic factor for HUVECs and HLECs, respectively, as demonstrated by enhanced tube formation. Ang2-induced hem- and lymphangiogenesis was mediated at least in part by SphK1 as shown by the inhibitory effect of SK1-I. Furthermore, both S1P and Ang2 significantly induced hem- and lymphangiogenesis in vivo determined by DIVAA/FACS. Similar to its effect on in vitro hem- and lymphangiogenesis, inclusion of SK1-I in the angioreactors together with Ang2 completely abolished its ability to stimulate both of these processes. CONCLUSION: Activation of SphK1 is necessary for Ang2-induced hemangiogenesis and lymphangiogenesis through the SphK1/S1P and Ang2/Tie2 axes. This work was supported by NIH (K12HD055881) and Susan G. Komen for the Cure (KG090510) to KT, NCI (R01CA61774) to SS, and Sumitomo Life Social Welfare Services Foundation grant to MN. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5280. doi:1538-7445.AM2012-5280