Decorin inhibits angiogenic potential of choroid-retinal endothelial cells by downregulating hypoxia-induced Met, Rac1, HIF-1α and VEGF expression in cocultured retinal pigment epithelial cells

Abstract

Choroidal neovascularization (CNV) is one of the most common causes of severe vision loss. Decorin, a multiple receptor tyrosine kinase inhibitor, has been recently shown to play an important regulatory role in angiogenic response. This study aims to investigate whether the overexpression of decorin in retinal pigment epithelial (RPE) cells under hypoxia alters the in vitro angiogenic ability of cocultured choroid-retinal endothelial cells and to explore the possible mechanisms involved. Human RPE cells (ARPE-19) were subjected to hypoxia with or without decorin pretreatment, and RNA interference technique was used to knock down the Met gene in ARPE-19 cells. Cell viability was determined using the Cell Counting Kit-8 assay. Expression of Met, Rac1 and hypoxia-inducible factor-1 alpha (HIF-1α) was evaluated by western blot and quantitative real-time polymerase chain reaction (qRT-PCR). Vascular endothelial growth factor (VEGF) expression was evaluated by enzyme-linked immunosorbent assay (ELISA) and qRT-PCR. We then constructed a recombinant lentiviral vector carrying the decorin gene to transduce ARPE-19 cells. The overexpression of decorin in transduced RPE cells was confirmed by qRT-PCR and western blot. The transduced RPE cells were then cocultured with rhesus macaque choroid-retinal endothelial cells (RF/6A) in a transwell coculture system to observe the effects of decorin overexpression in ARPE-19 cells on the proliferation, migration and tube formation of RF/6A cells. In response to hypoxia, the VEGF concentrations in the culture supernatants increased greatly at 24 and 48 h, and this effect was inhibited significantly and nearly equally in the presence of 50-200 nM decorin. Decorin pretreatment before hypoxia exposure effectively reduced the hypoxia-induced expression of Met, Rac1, HIF-1α and VEGF in ARPE-19 cells. Transfection of small interfering RNA against Met to ARPE-19 cells also resulted in significant downregulation of Rac1, HIF-1α and VEGF under hypoxia, and this effect was similar to that noted with decorin pretreatment alone or with their combination. Results from the coculture system showed that the overexpression of decorin in ARPE-19 cells significantly inhibited the proliferation, migration and tube formation of RF/6A cells. These results indicate that Met pathway activation plays an important role in the upregulation of VEGF in RPE cells under hypoxia. Decorin may interfere with angiogenesis by downregulating hypoxia-induced Met, Rac1, HIF-1α and VEGF expression in RPE cells, which suggests a potential strategy for the inhibition of CNV.

Keywords: Met; choroidal neovascularization; decorin; hypoxia; retinal pigment epithelial cells; vascular endothelial growth factor.