FGFR-1-mediated Endothelial Cell Proliferation And Angiogenesis Promoted By Vaccarin

Vaccarin is the main active component in Semen Vaccariae.At present,the known activity of vaccarin is primarily its effects on endothelial cells.For example,by inhibiting the Notch signaling pathway,apoptosis of human EA hy926 endothelial cells induced by H2O2 can be prevented by vaccarin.The proliferation,migration and tube formation of HMEC-1 endothelial cells can be stimulated by vaccarin in vitro,and angiogenesis in vivo can be accelerated by Vaccarin.However,the target receptor and related mechanism of the effect that vaccarin can promote HMEC-1 endothelial cells proliferation and angiogenesis are not clear.In this study,the target receptor and related mechanism of the effect that vaccarin could promote endothelial cells proliferation and angiogenesis would be investigated in depth.The activity of vaccarin on the promotion of endothelial cell proliferation was studied: In the SRB experiment,vaccarin significantly promoted the proliferation of HMEC-1 and EA hy926 cells,and this promotion was non-dose-dependent with the optimal concentration of 2.15μM.However,at the concentration of 2.15μM,vaccarin did not promote the proliferation of NIH3T3,A549 and RAW264.7 non-endothelial cells;In the trypan blue staining and Hoechst33342 staining expreriments,vaccarin did not significantly promoted HMEC-1 endothelial cells death and apoptosis;By flow cytometry,vaccarin could significantly promote HMEC-1 endothelial cells from G0/G1 phase to S phase,and this promotion was non-dosedependent with the optimal concentration of 2.15μM.Further,the target receptor in endothelial cells proliferation promoted by vaccarin was investigated: Western blotting showed that vaccarin had no significant effect on the phosphorylation of VEGFR-2.But vaccarin promoted the phosphorylation of FGFR-1 and its downstream signaling molecule ERK1/2.After blocking FGFR-1 by SU5402,the vaccarininduced phosphorylations of FGFR-1 and its downstream signaling molecule ERK1/2 were significantly inhibited;In the SRB experiment,the effect of vaccarin on endothelial cells proliferation was significantly inhibited after blocking FGFR-1.Further,the signaling pathway of endothelial cell proliferation promoted by vaccarin were studied;Western blotting showed that cyclinD1 was the downstream molecule of ERK1/2 in FGFR-1-mediated endothelial cell proliferation signaling pathway,which was activated by vaccarin.Meanwhile,vaccarin could significantly activate the FGFR-1 signaling pathway of HMEC-1 and EA hy926 endothelial cells but could not significantly activate the FGFR-1 signaling pathway of the three nonendothelial cells of NIH3T3,A549 and RAW264.7;Immunofluorescence also showed that vaccarin could significantly activate FGFR-1 in HMEC-1 and EA hy926 endothelial cells but could not significantly activate FGFR-1 in the three non-endothelial cells of NIH3T3,A549 and RAW264.7;After removing FGF-2 in HMEC-1 cell culture medium by FGF-2 antibody,vaccarin-induced proliferation of endothelial cells was significantly inhibited.It indicated that vaccarin indirectly played a role in promoting endothelial cells proliferation through FGF-2;By ELISA,vaccarin could significantly increase the content of FGF-2 in HMEC-1 cell culture medium,and this increase was non-dose-dependent with the optimal concentration of 2.15μM.Meanwhile,vaccarin could also significantly increase the content of FGF-2 in EA hy926 cell culture medium at the concentration of 2.15μM,but it could not significantly improve the content of FGF-2 in the cell culture mediums of NIH3T3,A549 and RAW264.7.The activity of vaccarin on promoting angiogenesis was studied: Vaccarin was able to promote endothelial cells migration and tube formation in vitro;Vaccarin could promote angiogenesis in the Matrigels implanted in mice.Further,the mechanism of vaccarin on angiogenesis was studied: The effects of vaccarin on the migration and tube formation of endothelial cells were significantly inhibited after blocking FGFR-1 in vitro;The effect of vaccarin on the angiogenesis in the Matrigels implanted in mice was significantly inhibited after blocking FGFR-1 in vivo;Experiments in vitro revealed that vaccarin indirectly promoted the migration and tube formation of endothelial cells through FGF-2.In summary,vaccarin promoted endothelial cells proliferation and angiogenesis by activating the FGFR-1 signaling pathway.