L. J. Fernandez1, W. Huang1, K. P. Terracina1, A. Yamada5, T. Aoyagi3, S. Spiegel2, M. Nagahashi4, K. Takabe1,2 1Virginia Commonwealth University,Surgical Oncology,Richmond, VA, USA 2Virginia Commonwealth University,Biochemistry And Molecular Biology,Richmond, VA, USA 3Chiba University,Surgery,Chiba, , Japan 4Nigata University,Digestive And General Surgery,Nigata, , Japan 5Yokohama City University,Surgery,Yokohama, , Japan
Introduction:
It is well established from prior studies that both Angiopoietin2 (Ang2) and vascular endothelial growth factor-C (VEGF-C) in the human lymphatic endothelial cell (HLEC) play important roles in the induction of lymphangiogenesis in inflammation and cancer. Ang2 acts by activating the Tie-2 receptor and VEGF-C through VEGF receptor 3 (VEGFR-3). Sphingosine-1-phosphate (S1P), a lipid mediator, has emerged as an important player in cancer progression that promotes cell proliferation, survival, migration, angiogenesis and lymphangiogenesis. S1P is generated inside cells by sphingosine kinase 1 (SphK1), which is exported and exerts its functions by binding to its specific G protein coupled receptors. In HLECs, S1P binds to S1PR1 and causes lymphangiogenesis. To date, evidence of cross-talk between these pathways has not been reported
Methods:
HLECs were obtained from LONZA, and used after less than 4 splitting-passages. Protein levels of Ang2 and VEGF-C in the supernatant of cell culture were measured by ELISA, mRNA expression by qPCR, and phosphorylation was detected by Western Blot assay. SphK1 activity assay was used to compare the activity after treatment with Ang2 or VEGF-C
Results:
We found that both Ang2 and VEGF-C mRNA expression increased by S1P treatment, with a corresponding significant increase in VEGF-C and Ang2 in the supernatant of cell culture after S1P treatment. This implies that S1P promotes Ang2 and VEGF-C production and secretion from HLECs. Furthermore, with Western-blot we found that Tie2 was phosphorylated after S1P treatment as was VEGFR-3, which implies that Ang2 and VEGF-C bind to their receptors after S1P-induced secretion. We found that Ang-2 treatment to HLEC cells not only resulted in phosphorylated Tie-2 but also in phosphorylated SphK1. This result was similar with VEGF-C treatment which resulting not only VEGFR-3 phosphorylation but also in phosphorylated SphK1. This result is in agreement with that of an S1P activity assay that showed a 1.5 fold increase in SphK1 activity when HLEC were treated by Ang2 or VEGF-C when compared to control. These findings imply that both Ang2 and VEGF-C activate SphK1, which we know produces S1P, via activation of their receptors. Together with our previously published findings, we found that there is an amplification loop type cross-talk between S1P signaling and Ang2 and VEGF-C signaling in LECs
Conclusion:
Our results suggest that there is a feed-forward amplification loop in HLEC where S1P increases the levels and activities of both Ang2 and VEGF-C, then in turn, Ang2 and VGEF-C activate SphK1 that produces S1P. S1P as well as Ang2 and VEGF-C have been independently implicated to be involved in lymphangiogenesis. The feed-forward relationship between these previously separate pathways, will likely potentiate lymphangiogenesis when these pathways are activated. This may have important ramifications in the pathogenesis and treatment of inflammatory and neoplastic conditions