Synergistic Cytokine Signaling Drives Angiofibrotic Gene Pathways in Primary Human Retinal Endothelial Cells

Neovascularization of the posterior eye is a progressive disease state marked by inflammatory initiation, angiogenic growth, and subsequent fibrotic degeneration of endothelial cells (ECs). Although a leading cause of irreversible vision loss worldwide, implicated in age-related macular degeneration and proliferative diabetic retinopathy, the mechanisms underpinning retinal EC dysregulation in neovascularization and fibrosis remain poorly understood. This study presents a transcriptomic investigation of primary cultured human microvascular retinal EC dysregulation exposed for 24 hours to 10 ng/mL of six retinal neovascularization-associated signaling molecules [IL-6, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, TGF-β2, thrombin, and vascular endothelial growth factor-A], individually and in combination. TNF-α, thrombin, and TGF-β2 alone significantly enhanced expression of inflammatory and angiofibrotic pathways, including phosphatidylinositol 3-kinase/Akt, nuclear factor-κB, and SMAD signaling. BGN, CD34, COL1A2, CXCL8, IGFBP5, INHBA, SERPINE1, SNAI1, TGFB2, and TNFSF11 were identified as overlapping nodal genes in retinal EC dysfunction. Cotreatment with all six signaling molecules significantly enhanced differential expression, revealing 889 unique differentially expressed genes. The novel network-based gene correlation engine, GeneBunny, revealed the cotreated group more accurately recapitulated published age-related macular degeneration patient-derived retinal and choroidal EC transcriptomes more accurately than any single-ligand treated group. These findings provide a biologically relevant characterization of retinal EC pathology driven by key retinal neovascularization-associated signaling molecules, enabling the identification of novel anti-fibrotic therapeutic targets.