Sphingosine-1-phosphate의 림프관 신생 및 박테리아 유래 세포밖 소포체의 혈관 신생 조절 활성 연구

Abstract

The vascular system plays important roles in supplying fresh oxygen and nutrients to tissues while the lymphatic system plays pivotal roles in mediating tissue fluid homeostasis and immunity. Furthermore, excessive blood and lymphatic vessel formation is implicated in many pathological conditions, which include inflammation and tumor metastasis. Tumor cells metastasize to other distant organs via newly formed blood and lymphatic vessels. Therefore, angiogenesis, lymphangiogenesis, and lymph node metastasis are very important diagnostic indicator to predict tumor progression. However, although several kinds of angiogenic factors are known to regulate lymphangiogenesis, the molecular mechanisms that regulate lymphatic vessel formation remain poorly characterized. Sphingosine-1-phosphate (S1P) is a potent bioactive lipid that is implicated in a variety of biological processes such as inflammatory responses and angiogenesis. In this study, it is revealed that S1P acts as a lymphangiogenic mediator. S1P induced migration, capillary-like tube formation, and intracellular Ca2+ mobilization, but not proliferation, in human lymphatic endothelial cells (HLECs) in vitro. Moreover, a Matrigel plug assay demonstrated that S1P promoted the outgrowth of new functional lymphatic vessels in vivo. HLECs expressed S1P1 and S1P3, and both RNA interference-mediated down regulation of S1P1 and an S1P1 antagonist significantly blocked S1P-mediated lymphangiogenesis. Furthermore, pertussis toxin, U73122, and BAPTA-AM efficiently blocked S1P-induced in vitro lymphangiogenesis and intracellular Ca2+ mobilization of HLECs, indicating that S1P promotes lymphangiogenesis by stimulating S1P1/Gi/phospholipase C/Ca2+ signaling pathways. These results suggest that S1P is the first lymphangiogenic bioactive lipid to be identified, and that S1P and its receptors might serve as new therapeutic targets against inflammatory diseases and lymphatic metastasis in tumors. Intercellular communications, from prokaryotes to eukaryotes, are essential process in living organisms, and are believed to be mediated by the soluble factors, cell-to-cell contacts, and the formation of nanotubes between the cells. Recently, membrane vesicles have drawn much attention to one of communicators. Membrane vesicles derived from bacteria are called as outer membrane vesicles (OMVs) and consist of outer membrane proteins, lipopolysaccharide, outer membrane lipids, and DNA/RNA. Bacterial OMVs have been known to play pivotal roles in the delivery of toxins to host cells, in the transfer of proteins and genetic materials between bacteria, in interspecies signaling, and in the elimination of competing microorganisms. Although there have been studies on the roles of lipopolysaccharide, DNA, and bacteria on angiogenesis, however, there were no approaches to prove the effects of bacterial OMVs on angiogenesis. In this study, it is proved that Escherichia coli C4 OMVs promote angiogenesis by inducing the release of IL-6 from alternatively activated macrophages. E. coli C4 OMVs induced in vivo angiogenesis in a Matrigel plug assay, but showed no direct effects on the migration, proliferation, and tube formation of blood endothelial cells. However, E. coli C4 OMVs promoted the infiltration of macrophages into Matrigels, suggesting indirect effects of E. coli C4 OMVs on endothelial cells via macrophage activation. Conditioned medium harvested from E. coli C4 OMVs-stimulated macrophages induced the migration of endothelial cells. In addition, E. coli C4 OMVs induced the release of IL-6, TNF-α, and IL-10 and reduced the production of IL-12p40 from peritoneal macrophages, showing similar cytokine secretion patterns to alternatively activated M2b type macrophages. Furthermore, E. coli C4 OMVs could not induce in vivo angiogesis and in vitro endothelial cell migration in IL-6-/- mice, indicating that IL-6 is important to OMVs-mediated angiogenesis. These results suggest that bacterial OMVs are important to bacteria survival and growth in host by suppressing immune responses and promoting angiogenesis via alternatively activated macrophages.