BackgroundAge-related macular degeneration (AMD) is a severe eye disease that affects the macula, the central area of the retina. Current treatment consists of intravitreal injections of vascular endothelial growth factor (VEGF) inhibitors. However, some patients remain refractory to treatment even after maximal anti-VEGF dosage. We longitudinally analyzed angiogenic and inflammatory biomarkers in the aqueous humor (AH) of these patients.Objectives1) To confirm the presence of angiogenic and inflammatory biomarkers before (in the naive state) and after two anti-VEGF injections; and 2) to characterize the inflammatory response according to the type of anti-VEGF used (ranibizumab, aflibercept, brolucizumab, or faricimab).MethodsAH samples were collected from two groups of patients: 35 patients with AMD and 17 control patients with no retinopathy. Eleven of the 35 patients with AMD were characterized as refractory (rAMD) after long-term anti-VEGF treatment. Multiplex and proximity extension assays (PEA) were performed on these samples to define their inflammatory profiles. Free VEGFA was detected using an additional AlphaLISA VEGFA assay.ResultsSeveral inflammatory molecules are upregulated in AMD, while a smaller subset is downregulated in rAMD, indicating that these could serve as markers of a refractory state. In contrast, no differences were observed in the biomarkers previously reported as increased in rAMD after long term treatment. These findings suggest that anti-VEGF treatment induces the expression of angiogenic and inflammatory biomarkers. Analysis of the inflammatory response following the injection of different anti-VEGF agents revealed exacerbated inflammation in patients treated with brolucizumab, whereas ranibizumab caused the lowest inflammatory response. In addition, we compared three different assays to assess AH VEGFA, but only the AlphaLISA assay could detect free VEGFA.ConclusionOur results indicate that anti-VEGFA therapy shapes AMD-associated inflammation; therefore, the choice of anti-VEGFA therapy remains important in reducing secondary inflammatory effects. Furthermore, the detection of free VEGF is dependent on the assay used.