The Tg (fli1: egfp; gata1: dsred) transgenic zebrafish embryos were treated with different concentrations of PNS (10, 50, 100 μM) for 48 h with or without the 6 h pretreatment of the 30 μM Vascular endothelial growth factors receptor (VEGFR)-3 kinase inhibitor, followed with morphological observation and lympangiogenesis of thoracic duct assessment. The effect of PNS on cell viability, migration, tube formation and Vascular endothelial growth factors (VEGF)-C mRNA and protein expression of lymphatic endothelial cells (LECs) were determined. The role of phosphatidylinositol-3 (PI-3)-kinase (PI3K), extracellular signal-regulated kinase (ERK)1/2 pathways, c-Jun N-terminal kinase (JNK) and P38 mitogen activated protein kinases (MAPK) signaling in PNS-induced VEGF-C expression of LECs by using pharmacological agents to block each signal.
PNS promotes lymphangiogenesis of thoracic duct in zebrafish with or without VEGFR3 Kinase inhibitor pre-impairment. PNS promotes proliferation, migration and tube formation of LECs. The tube formation induced by PNS could be blocked by VEGFR3 Kinase inhibitor. PNS induce VEGF-C expression of LEC, which could be blocked by ERK1/2, PI3K and P38MAPK signaling inhibitors.
PNS activates lymphangiogenesis both in vivo and in vitro by up-regulating VEGF-C expression and activation of ERK1/2, PI3K and P38MAPK signaling. These findings provide a novel insight into the role of PNS in lymphangiogenesis and suggest that it might be an attractive and suitable therapeutic agent for treating secondary lymphedema or other lymphatic system impairment related disease.