Authors: Wu Jing, Liu Gaoqin, Chen Zhigang, Xiao Yanhui, Xu Jing, Lu Peirong
Researches showed that ADP-ribosylation factor (ARF) promotes intracorneal secretion of multiple angiogenesis-related factors, such as vascular endothelial growth factor (VEGF) and nitricoxide synthase (NOS) etc., and therefore results in corneal neovascularization.However, whether ARF affects the tube formation of human retinal endothelial cells(HRECs) is unelucidated.Understanding the effect of ARF tube formation of HRECs is important for the target treatment of retinal vascular diseases.
The aim of this study was to explore the effect and mechanism of ARF inhibitor on tube formation of HRECs in vitro.
HRECs (HREC line) were cultured and passaged.The growth-well cells were harvested and divided into two groups.The cells were regularly cultured in the control group, and ARF antagonist (1ml) was added in the culture medium in the ARF antagonist group.The expression levels of ARFmRNA and protein in the cells were examined by reverse transcription (RT)-PCR and Western blot.The morphology and number of HREC tube formation were detected by using three-dimensional Matrigel assay.The relative expression levels of VEGF, NOS, focal adhesion kinase (FAK) and heat shock protein 90 (HSP90) at gene level and protein level were examined by RT-PCR and Western blot in vitro.
The relative expression levels of ARFmRNA in the cells were 0.65±0.14 and 0.32±0.10, and those of ARF protein were 0.85±0.15 and 0.24±0.17 in the control group and ARF antagonist group, showing significant differnces between the two groups (t=7.32, P=0.00; t=5.15, P=0.00). The number of HREC tube formation was (34.66±8.57)/field in the ARF antagonist group, which was significantly lower than (51.46±7.12)/field in the control group (t=2.99, P=0.04). The relative expression levels of VEGF mRNA, NOSmRNA and their proteins in the cells were significantly lower than those of the control group (t=3.02, P=0.04; t=3.68, P=0.02; t=3.33, P=0.03; t=2.89, P=0.04). The relative expression levels of FAKmRNA and HSP90mRNA in the ARF antagonist group were 0.65±0.18 and 0.28±0.05, which were significantly lower than 0.76±0.25 and 0.46±0.09 in the control group (all at P<0.05).
ARF antagonist appears to have an inhibitory effect on the tube formation ability of HRECs propably by down-regulating the expressions of VEGF, NOS and the downstream signal transduction factors FAK and HSP90 in HRECs in vitro.