IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

Abstract

Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) has been reported to have various functions in different cellular contexts. Our previous investigation discovered that IGFBP-rP1 inhibited retinal angiogenesis in vitro and in vivo by inhibiting the pro-angiogenic effect of VEGF and downregulating VEGF expression. Recently, IGFBP-rP1 was confirmed to be downregulated in the aqueous humor of patients with neovascular age-related macular degeneration compared with controls; however, its specific role remains unknown. The present study applied the technique of gene silencing, reverse transcription-quantitative PCR, western blotting, cell viability assays, cell motility assays and tube formation assays. Chemical hypoxic conditions and choroidal endothelial (RF/6A) cells were used to explore the effect of IGFBP-rP1-silencing on the phenotype activation of RF/6A cells under hypoxic conditions and to elucidate the underlying mechanisms. siRNA achieved IGFBP-rP1-silencing in RF/6A cells without cytotoxicity. IGFBP-rP1-silencing significantly restored the viability of RF/6A cells in hypoxia and enhanced hypoxia-induced migration and capillary-like tube formation of RF/6A cells. Furthermore, IGFBP-rP1-silencing significantly upregulated the expression of B-RA F, phosphorylated (p)-MEK, p-ER K and VEGF in RF/6A cells under hypoxic conditions; however, these upregulations were inhibited by exogenous IGFBP-rP1. These data indicated that silencing IGFBP-rP1 expression in RF/6A cells effectively promoted the hypoxia-induced angiogenic potential of choroidal endothelial cells by upregulating RA F/MEK/ER K signaling pathway activation and VEGF expression.