Results Tylophorine inhibited cell viability in endothelial cells Angiogenesis is primarily initiated Cisplatin by growth factors there fore we tested whether tylophorine decreases VEGF mediated HUVEC viability and proliferation. We found that when HUVECs were cultured in normal cell culture medium in absence of VEGF, tylophorine inhibited cell viability in a dose and time dependent manner. Significant cell viability inhibitory effect of tylophorine was observed in HUVECs at concen trations more than 10 uM. As shown in Figure 1C, the proliferation of endothelial cells stimulated by VEGF was markedly decreased after tylophorine treat ment ranging from 2. 5 to 20 uM at different time intervals of 24 and 48 h indicating extracellular VEGF acted as a strong attractant for endothelial cells proliferation.

Tylophorine alone inhibited the growth of HUVEC in dose dependent manner. As detected by BrdU incorporation assay, DNA synthesis of HUVECs was also significantly Inhibitors,Modulators,Libraries inhibited by tylophorine Inhibitors,Modulators,Libraries in a dose dependent manner. To further exam ine whether tylophorine would result in toxic effects of HUVEC, LDH cytotoxic assay was carried out. As shown in Figure 1E, Tylophorine caused minute toxicity on HUVECs. Tylophorine inhibited VEGF induced endothelial cell migration and invasion and tube formation of HUVECs Cell migration is an essential step in angiogenesis. therefore we investigated the effects of tylophorine on the chemotactic motility of the endothelial cells by using wound healing assay. The results showed that tylophorine significantly inhibited VEGF induced HUVECs migration in a dose dependent manner ran ging from 2.

5 uM to 20 uM. Directional Inhibitors,Modulators,Libraries motility and matrix degradation are crucial for angiogenesis sprouting therefore, we next examined the effect of tylophorine on the invasion ability of HUVECs using the Boyden chamber assay. As shown in Figure 2B, a large Inhibitors,Modulators,Libraries number of cells migrated to the lower side Inhibitors,Modulators,Libraries of membrane in the transwell chamber after stimulation with VEGF. How ever, the number of invaded cells were significantly low in the presence of tylophorine. The matur ation of migrated endothelial cells into a capillary tube is a critical step during angiogenesis. Thus, we investi gated its effect on HUVEC tube formation. When HUVECs were seeded on the growth factor reduced matrigel, robust tubular like structures were formed in the presence of VEGF.

Almost 80% destruc tion of tube network was observed when HUVECs were incubated with tylophorine at 10 uM. Taken together, tylophorine suppressed VEGF induced angio genesis in vitro by inhibiting the migration, invasion and tubular structure formation of endothelial cells. Differential effect of tylophorine on the binding of VEGF to its receptors Further, we investigated enough whether tylophorine inhibits the binding of VEGF to its receptors, VEGFR1 and VEGFR2.