Effect of Cathepsin B and vascular endothelial growth factor on the retinal neovascularization induced by hyperoxia

Background

Vascular endothelial growth factor (VEGF) plays a key role in the vascular development and neovascularization.Studies have proved that Cathepsin B is related to the formation of neovascularization, but its mechanism is unclear.

Objective

This study was to investigate the expression of Cathepsin B and VEGF in the retinal neovascularization induced by hyperoxia and the relationship between them.

Methods

Forty-four 7-day-old C57BL/6J mice were randomly assigned into 4 groups: normal control group, hyperoxia-induced group, normal control (NC)-green fluorescent protein (GFP)-lentivirus (Lv) group and Cathepsin B-RNA interference (RNAi)-Lv group, with 11 mice 22 eyes for each group.The mice in normal control group were survival in natural environment.The other three groups of 7-day-old mice were put in a sealed box of oxygen volume fraction (75±2)% for 5 days and then sent back to normal environment.The mice of hyperoxia-induced group did not have any drug intervention, while the 12-day-old mice of NC-GFP-Lv group and Cathepsin B-RNAi-Lv group received intravitreal injection of NC-GFP-Lv 1 μl or Cathepsin B-RNAi-Lv 1 μl.All 17-day-old mice were sacrificed and the retinas were collected.The mRNA expression levels of Cathepsin B and VEGF were performed by real-time PCR; the protein expression levels of Cathepsin B and VEGF were detected by Western blot.

Results

Fluorescence microscope results showed that the layer and branch of retinal neovascularization were less in the Cathepsin B-RNAi-Lv group than those in the NC-GFP-Lv group and hyperoxia-induced group.The relative expression levels of Cathepsin B and VEGF mRNA in each group were significantly different (F=444.89, P=0.00; F=519.78, P=0.00). The relative expression levels of Cathepsin B and VEGF mRNA in hyperoxia-induced group, NC-GFP-Lv group and Cathepsin B-RNAi-Lv group were higher than those in normal control group, and the relative expression levels of Cathepsin B and VEGF mRNA in Cathepsin B-RNAi-Lv group were lower than those in hyperoxia-induced group and NC-GFP-Lv group, with significant differences between them (all at P<0.05). The relative expression levels of Cathepsin B and VEGF protein in each group were significantly different (F=54.37, P=0.00; F=79.65, P=0.00). The relative expression levels of Cathepsin B and VEGF protein in hyperoxia-induced group, NC-GFP-Lv group and Cathepsin B-RNAi-Lv group were higher than those in normal control group, and the relative expression levels of Cathepsin B and VEGF protein in Cathepsin B-RNAi-Lv group were lower than those in hyperoxia-induced group and NC-GFP-Lv group, with significant differences between them (all at P<0.05).

Conclusions

The expression levels of Cathepsin B and VEGF in retinal neovascularization of hyperoxia-induced group were significantly higher than those in normal control group, Cathepsin B-RNAi-Lv can inhibit the mRNA and protein expression of Cathepsin B and VEGF, the expression of VEGF may be influenced by the expression of Cathepsin B.