Inhibitor of growth 4 affects hypoxia-induced migration and angiogenesis regulation in retinal pigment epithelial cells.

Abstract

Inhibitor of growth 4 (ING4), a potential tumor suppressor, is implicated in cell migration and angiogenesis. However, its effects on diabetic retinopathy (DR) have not been elucidated. In this study, we aimed to evaluate ING4 expression in normal and diabetic rats and clarify its effects on hypoxia-induced dysfunction in human retinal pigment epithelial (ARPE-19) cells. A Type 1 diabetic model was generated by injecting rats intraperitoneally with streptozotocin and then killed them 4, 8, or 12 weeks later. ING4 expression in retinal tissue was detected using western blot analysis, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), and immunohistochemistry assays. After transfection with an ING4 overexpression lentiviral vector or small interfering RNA (siRNA), ARPE-19 migration under hypoxia was tested using wound healing and transwell assays. The angiogenic effect of conditioned medium (CM) from ARPE-19 cells was examined by assessing human retinal endothelial cell (HREC) capillary tube formation. Additionally, western blot analysis and RT-qPCR were performed to investigate the signaling pathways in which ING4, specificity protein 1 (Sp1), matrix metalloproteinase 2 (MMP-2), MMP-9, and vascular endothelial growth factor A (VEGF-A) were involved. Here, we found that ING4 expression was significantly reduced in the diabetic rats' retinal tissue. Silencing ING4 aggravated hypoxia-induced ARPE-19 cell migration. CM collected from ING4 siRNA-transfected ARPE-19 cells under hypoxia promoted HREC angiogenesis. These effects were reversed by ING4 overexpression. Furthermore, ING4 suppressed MMP-2, MMP-9, and VEGF-A expression in an Sp1-dependent manner in hypoxia-conditioned ARPE-19 cells. Overall, our results provide valuable mechanistic insights into the protective effects of ING4 on hypoxia-induced migration and angiogenesis regulation in ARPE-19 cells. Restoring ING4 may be a novel strategy for treating DR.