Macrophage extracellular traps amplify retinal endothelial anoikis via the S1P–S1PR axis in diabetic retinopathy

OBJECTIVE: To investigate the pathogenic role of macrophage extracellular traps (METs) in proliferative diabetic retinopathy (PDR), focusing on endothelial dysfunction and inflammatory activation. METHODS: Transcriptomic data from PDR patients were first analyzed to identify METs-associated pathways. Anoikis-related subgroups were defined using the median gene set variation analysis (GSVA) enrichment score of the anoikis gene set, and enriched pathways were selected for subsequent validation. In vitro, diabetic retinopathy models were established using high-glucose and METs stimulation. Human retinal microvascular endothelial cells (HRMECs) were assessed for functional alterations and apoptosis. Rescue assays employed the AKT agonist SC79, the endocytosis inhibitor Dynasore, and DNase I. In vivo, streptozotocin (STZ)-induced diabetic mice received intravitreal METs with or without pharmacological interventions to evaluate vascular leakage, inflammatory responses, and neovascularization. RESULTS: Transcriptomic analysis revealed a strong association between METs-induced endothelial injury and activation of anoikis pathways, with sphingolipid signaling significantly enriched in the anoikis-high subgroup. In vitro, METs disrupted endothelial barrier integrity, induced ROS accumulation and mitochondrial damage, and activated anoikis and inflammatory signaling, accompanied by FAK dephosphorylation. These effects were partially reversed by SC79, DNase I, and sphingolipid-pathway inhibition. METs were internalized by HRMECs via endocytosis, triggering downstream signaling. In vivo, intravitreal METs induced vascular leakage, inflammatory cytokine elevation, and neovascularization, whereas inhibition of the SPHK1/S1P pathway (SKI-II) significantly mitigated these pathological changes. CONCLUSION: METs promote retinal vascular dysfunction by inducing endothelial anoikis and inflammatory activation through FAK/AKT and SPHK1/S1P/S1PR2/NF-κB signaling. Targeting METs-triggered lipid signaling may offer new therapeutic insights for diabetic retinopathy.