Regulation of axon guidance cue netrin-1 on angiogenesis

Abstract

To study the effect of netrin-1, an axon guidance cue, on angiogenesis. Human umbilical cord vein endothelial cells (HUVECs) were isolated and cultured. Reverse transcription and polymerase chain reaction (RT-PCR) was used to detect all the known receptors of netrin-1 in the HUVECs. Netrin-1 and vascular endothelial growth factor (VEGF) of the concentration of 10 ng/ml were added into the culture fluid respectively for 72 h, cholecystokinin-8 (CCK-8) was added, and then enzyme mark instrument was used to calculate the relative absorbance (A value). Other HUVECs were added into the upper chamber of the Transwell co-culture system and netrin-1 of different concentrations and VEGF of the concentration of 10 ng/ml were added into the lower chamber respectively for 6 h, and then invert microscopy were used to observe the migration of HUVECs. Matrigel was added into the 96-well plate, and HUVECs and netrin-1 of different concentrations were added into the wells, then contrast microscopy was used to calculate the tube formation. Corneal micropocket assay was performed on 96 rabbits to determine the corneal neovascularization (CNV) with treatment with different concentrations of netrin-1. Of the 6 ligands only the receptor UNC5B was expressed in the HUVECs. The cell counting kit-8 assay showed that the proliferation of HUVECs was promoted when the concentration of netrin-1 was under 500 ng/ml, especially when the concentration was 50 ng/ml, however, when the netrin-1 concentrations were 1000 to 5000 ng/ml the proliferation of the HUVECs was inhibited (P<0.05). The migration of HUVECs was promoted when the concentration of netrin-1 was under 500 ng/ml, especially when the concentration was 100 ng/ml, however, when the netrin-1 concentrations were 1000 to 5000 ng/ml the migration of the HUVECs was inhibited (P<0.05). Normally HUVECs formed branch-like and tube-like structure in the culture plate, netrin-1 did not influence the tube formation when the concentration was under 500 ng/ml, and inhibited the tube formation when the concentration was 1000 approximately 5000 ng/ml (P<0.05). Rabbit corneal micropocket assay showed that netrin-1 of the concentration of 100 ng/ml and VEGF of the concentration of 10 ng/ml promoted the angiogenesis, however, the netrin-1 of the concentration of 5000 ng/ml inhibited the angiogenesis. Netrin-1 shows has a dual function, both promotive or inhibitory effects, on angiogenesis, depending on the concentration and its inhibitive effect is mediated by UNC5B.