AIF-1 Drives Corneal Neovascularization by Promoting Inflammatory Macrophage Activation via the MAPK and PI3K/AKT/mTOR Signaling Pathways.

Abstract

PURPOSE: This study aims to elucidate the precise mechanisms of allograft inflammatory factor 1 (AIF-1)-mediated inflammatory macrophage activation in corneal neovascularization (CNV) pathogenesis and identify a potential therapeutic target. METHODS: In vitro, RAW 264.7 macrophages were transfected with small interfering RNA (siRNA) targeting AIF-1. Flow cytometry and immunofluorescence were used to detect macrophage polarization, and ELISA was employed to quantify inflammatory cytokines secreted by macrophages. Intracellular reactive oxygen species (ROS) levels were measured using a ROS detection kit. In vivo, a mouse CNV model was established by alkali burns. AIF-1 siRNA was administered via subconjunctival injection. Inflammatory cytokines and angiogenesis-related factors in the cornea were assessed through Western blot (WB), immunohistochemistry, and immunofluorescence. Slit-lamp imaging and histopathologic analysis were conducted to evaluate angiogenesis and inflammation. Phosphorylation levels of proteins in the MAPK and PI3K/AKT/mTOR pathways were examined by WB. RESULTS: We observed stage-dependent upregulation of AIF-1, paralleled by CNV severity, alongside coordinated overexpression of CD86 and VEGFA. Silencing AIF-1 suppressed inflammatory macrophage activation. AIF-1 promoted macrophage recruitment to the cornea and upregulated TNF-α, IL-1β, IL-6, and VEGFA expression, further exacerbating inflammation, oxidative stress, and angiogenesis in the mouse CNV model. CNV severity was alleviated after AIF-1 inhibition. Notably, AIF-1 drove CNV progression by activating both the MAPK and PI3K/AKT/mTOR signaling pathways. CONCLUSIONS: AIF-1 played a pivotal role in CNV progression. AIF-1 promoted inflammatory macrophage activation, exacerbating corneal inflammation and neovascularization via the MAPK and PI3K/AKT/mTOR signaling pathways, which offers novel insights for CNV treatment.