Rabbit Anti-S1PR1 monoclonal antibody, clone KN21-77

Inflammation-induced lymphangiogenesis is a widely accepted concept. However, most of the inflammatory factors and their related mechanisms have not been clarified. It has been reported that sphingosine-1-phosphate (S1P) is not only closely related to the chronic inflammatory process but also affects angiogenesis. Therefore, we investigated the inflammatory effects of S1P on human lymphatic endothelial cells (HLECs). Our results showed that S1P promotes tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) secretion in HLECs. We also confirmed that S1P-stimulated TNF-alpha and IL-1 beta secretion is mediated through S1P receptor 1 (S1PR1). Using TNF-alpha SiRNA and IL-1 beta siRNA, we found that TNF-alpha and IL-1 beta play essential roles in S1P-induced HLEC proliferation, migration, and tube formation. Sip induces phosphorylation of NF-kappa B p65 and activation of NF-kappa B nuclear translocation. A S1PR1 antagonist (W146) and NF-kappa B inhibitor (BAY11-7082) inhibited S1P-induced TNF-alpha and IL-1 beta secretion and prevented NF-kappa B nuclear translocation. Taken together, the results demonstrated for the first time that S1P promotes the secretion of TNF-alpha and IL-1 beta in HLECs via S1PR1-mediated NF-kappa B signaling pathways, thus affecting lymphangiogenesis. The study provides a new strategy for finding treatments for lymphangiogenesis-related diseases.

Rationale: Modulation of pulmonary vascular barrier function is an important clinical goal given the devastating effects of vascular leak in acute lung injury (ALI). We previously demonstrated that FTY720 S-phosphonate (Tys), an analog of sphingosine 1-phosphate (S1P) and FTY720, has more potent pulmonary barrier protective effects than these agents in vitro and in mouse models of ALI. Tys preserves expression of the barrier-promoting S1P1 receptor (S1PR1), whereas S1P and FTY720 induce its ubiquitination and degradation. Here we further characterize the novel barrier promoting effects of Tys in cultured human pulmonary endothelial cells (EC). Methods/Results: In human lung EC, Tys significantly increased peripheral redistribution of adherens junction proteins VE-cadherin and beta-catenin and tight junction protein ZO-1. Inhibition of VE-cadherin with blocking antibody significantly attenuated Tys-induced transendothelial resistance (TER) elevation, while ZO-1 siRNA partially inhibited this elevation. Tys significantly increased focal adhesion formation and phosphorylation of focal adhesion kinase (FAK). Pharmacologic inhibition of FAK significantly attenuated Tys-induced TER elevation. Tys significantly increased phosphorylation and peripheral redistribution of the actin-binding protein, cortactin, while cortactin siRNA partially attenuated Tys-induced TER elevation. Although Tys significantly increased phosphorylation of Akt and GSK3 beta, neither PI3 kinase nor GSK3 beta inhibition altered Tys-induced TER elevation. Tys significantly increased Rac1 activity, while inhibition of Rac1 activity significantly attenuated Tys-induced VE-cadherin redistribution and TER elevation. Conclusion: Junctional complex, focal adhesion rearrangement and Rac1 activation play critical roles in Tys-mediated barrier protection in pulmonary EC. These results provide mechanistic insights into the effects of this potential ALI therapy. (C) 2015 Elsevier Inc. All rights reserved.