Angiopoietin-2 Exocytosis Is Stimulated by Sphingosine-1-Phosphate in Human Blood and Lymphatic Endothelial Cells

Abstract

Objective-Although diverse functions of angiopoietin-2 (Ang2) have been revealed, little is known about upstream signaling molecules regulating Ang2 exocytosis. We therefore investigated the mechanism of Ang2 exocytosis in human blood and lymphatic endothelial cells (BECs and LECs) by stimulation with sphingosine-1-phosphate (S1P). Methods and Results-By immunostaining and ELISA analyses using our newly developed human Ang2-specific antibodies, Ang2 exocytosis from human endothelial cells was examined. Both exogenous and endogenous S1P trigger rapid Ang2 exocytosis in time-and dose-dependent manners. Intriguingly, S1P-induced Ang2 exocytosis is higher in LECs than BECs. These effects of S1P are mainly mediated by the endothelial differentiation gene receptor 1, which subsequently activates its downstream phospholipase C and intracellular calcium mobilization to trigger Ang2 exocytosis. Consistently, S1P also dramatically stimulates Ang2 exocytosis from the ECs of ex vivo-incubated blood vessels. ngiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) were identified as key regulators of pathophysiological angiogenesis 1-4 along with vascular endothelial growth factors (VEGFs). Both Ang1 and Ang2 are secretory proteins and have an amino-terminal coiled-coil domain for their oligomerization and a carboxy-terminal fibrinogen-like domain (FLD) for binding to their receptor, Tie2. 1,2 In contrast to the consistent role of Ang1 in stabilizing newly-formed vessels, 1,4 more versatile functions have been reported for Ang2 such as angiogenesis, lymphangiogenesis, vascular permeability, and inflammation. 2,5-9 Intriguingly, Ang2 is synthesized in advance and stored in Weibel-Palade bodies (WPBs) of endothelial cells (ECs), thereby being rapidly secreted to the external microenvironment by exocytosis when stimulated by extracellular signals. 10,11 This implies that Ang2 activity is temporally regulated to exert controlled actions onto Tie2-expressing cells via autocrine and paracrine rather than endocrine manner. However, despite the importance of Ang2 exocytosis control, signaling pathways regulating Ang2 exocytosis are poorly understood. Conclusion-TheseLymphatic endothelial cells (LECs) have their own characteristic morphologies, functions, and specific gene expressions compared to blood endothelial cells (BECs). 12 Lymphatic vessels function as a key route for immune cell trafficking and interstitial fluid drainage, and directly participate in inflammation and tissue homeostasis. 13,14 Interestingly, Ang2 knockout mice exhibit defective lymphatic vessel development, 6 implicating essential role of Ang2 in development and function of LECs. Although the expression of Ang2 in LECs has been documented, 15,16 presence of Ang2 granules and regulation of Ang2 secretion are still unknown.Sphingosine-1-phosphate (S1P) is a phospholipid signaling molecule generated from the plasma-membrane sphingosine by sphingosine kinases 1 and 2. 17 S1P is mainly secreted from platelets, monocytes, and mast cells, thereby governing various cellular p...

Lymphangiogenic factors, mechanisms, and applications

“…In vitro, S1P induces LEC migration and tube formation via the S1P1/G i /PLC/Ca 2+ pathway (70,71). Moreover, S1P stimulates ANG2 secretion from LECs much more potently than from BECs (72). Given that ANG2 is required for lymphatic development (55,56), S1P may act synergistically with ANG2 in lymphangiogenesis.…”

Targeting the Angiopoietin-2/Tie-2 axis in conjunction with VEGF signal interference

“…Ang-2 is stored in Weibel–Palade bodies in endothelial cells and secreted upon microenvironmental changes [30–33]. Contrary to Ang-1/Tie2 signaling Ang-2 autocrine signaling via the Tie2 receptor results in loss of cell–cell contacts and vascular destabilization (Fig.…”

Regulation of Cellular Communication by Signaling Microdomains in the Blood Vessel Wall

“…The sphingolipid S1P is similar to thrombin and histamine in that it can potentiate inflammation in the vasculature via exocytosis of vWF and angiopoietin 2 from WPBs (Matsushita et al, 2004;Jang et al, 2009). Matsushita et al (2004) showed that S1P-mediated exocytosis of WPBs occurs in part via PLCg but also involves extracellular calcium, because both PLC inhibitors and calcium-free media blocked S1P-induced secretion of vWF from human aortic ECs.…”